Method of detecting target base sequence of rna interference, method of designing polynucleotide base sequence causing rna interference, method of constructing double-stranded polynucleotide, method of regulating gene expression, base sequence processing apparatus, program for running base sequence processing method on comp

ABSTRACT

In the present invention, a sequence segment conforming to the following rules (a) to (d) is searched from the base sequences of a target gene of RNA interference and, based on the search results, siRNA capable of causing RNAi is designed, synthesized, etc.: (a) The 3′ end base is adenine, thymine, or uracil, (b) The 5′ end base is guanine or cytosine, (c) A 7-base sequence from the 3′ end is rich in one or more types of bases selected from the group consisting of adenine, thymine, and uracil, and (d) The number of bases is within a range that allows RNA interference to occur without causing cytotoxicity.

TECHNICAL FIELD

The present invention relates to RNA interference and more particularly,for example, to a method for designing sequences of polynucleotides forcausing RNA interference, the method improving efficiency in testing,manufacturing, etc., in which RNA interference is used. Hereinafter, RNAinterference may also be referred to as “RNAi”.

The present invention further relates to a base sequence processingapparatus, a program for running a base sequence processing method on acomputer, a recording medium, and a base sequence processing system. Inparticular, the invention relates to a base sequence processingapparatus capable of efficiently selecting a base sequence from the basesequences of a target gene, which causes RNA interference in a targetgene, a program for running a base sequence processing method on acomputer, a recording medium, and a base sequence processing system.

BACKGROUND ART

RNA interference is a phenomenon of gene destruction whereindouble-stranded RNA comprising sense RNA and anti-sense RNA (hereinafteralso referred to as “dsRNA”) homologous to a specific region of a geneto be functionally inhibited, destructs the target gene by causinginterference in the homologous portion of mRNA which is a transcript ofthe target gene. RNA interference was first proposed in 1998 followingan experiment using nematodes. However, in mammals, when long dsRNA withabout 30 or more base pairs is introduced into cells, an interferonresponse is induced, and cell death occurs due to apoptosis. Therefore,it was difficult to apply the RNAi method to mammals.

On the other hand, it was demonstrated that RNA interference could occurin early stage mouse embryos and cultured mammalian cells, and it wasfound that the induction mechanism of RNA interference also existed inthe mammalian cells. At present, it has been demonstrated that shortdouble-stranded RNA with about 21 to 23 base pairs (short interferingRNA, siRNA) can induce RNA interference without exhibiting cytotoxicityeven in the mammalian cell system, and it has become possible to applythe RNAi method to mammals.

DISCLOSURE OF INVENTION

The RNAi method is a technique which is expected to have variousapplications. However, while dsRNA or siRNA that is homologous to aspecific region of a gene, exhibits an RNA interference effect in mostof the sequences in drosophila and nematodes, 70% to 80% of randomlyselected (21 base) siRNA do not exhibit an RNA interference effect inmammals. This poses a great problem when gene functional analysis iscarried out using the RNAi method in mammals.

Conventional designing of siRNA has greatly depended on the experiencesand sensory perceptions of the researcher or the like, and it has beendifficult to design siRNA actually exhibiting an RNA interference effectwith high probability. Other factors that prevent further research beingconducted on RNA interference and its various applications are highcosts and time consuming procedures required for carrying out an RNAsynthesis resulting in part from the unwanted synthesis of siRNA.

Under such circumstances, it is an object of the present invention toprovide a more efficient and simplified means for the RNAi method.

In order to achieve the above object, the present inventors have studieda technique for easily obtaining siRNA, which is one of the stepsrequiring the greatest effort, time, and cost when the RNAi method isused. In view of the fact that preparation of siRNA is a problemespecially in mammals, the present inventors have attempted to identifythe sequence regularity of siRNA effective for RNA interference usingmammalian cultured cell systems. As a result, it has been found thateffective siRNA sequences have certain regularity, and thereby, thepresent invention has been completed. Namely, the present invention isas described below.

[1] A method for searching a target base sequence of RNA interferencecomprising: searching a sequence segment, conforming to the followingrules (a) to (d), from the base sequences of a target gene for RNAinterference:

(a) The 3′ end base is adenine, thymine, or uracil,

(b) The 5′ end base is guanine or cytosine,

(c) A 7-base sequence from the 3′ end is rich in one or more types ofbases selected from the group consisting of adenine, thymine, anduracil, and

(d) The number of bases is within a range that allows RNA interferenceto occur without causing cytotoxicity.

[2] The method for searching the target base sequence according to item[1], wherein, in the rule (c), at least three bases among the sevenbases are one or more types of bases selected from the group consistingof adenine, thymine, and uracil.

[3] The method for searching the target base sequence according to item[1] or [2], wherein, in the rule (d), the number of bases is 13 to 28.

[4] A method for designing a base sequence of a polynucleotide forcausing RNA interference comprising: searching a base sequence,conforming to the rules (a) to (d) below, from the base sequences of atarget gene and designing a base sequence homologous to the searchedbase sequence:

(a) The 3′ end base is adenine, thymine, or uracil,

(b) The 5′ end base is guanine or cytosine,

(c) A 7-base sequence from the 3′ end is rich in one or more types ofbases selected from the group consisting of adenine, thymine, anduracil, and

(d) The number of bases is within a range that allows RNA interferenceto occur without causing cytotoxicity.

[5] The method for designing the base sequence according to item [4],wherein, in the rule (c), at least three bases among the seven bases areone or more types of bases selected from the group consisting ofadenine, thymine, and uracil.

[6] The method for designing the base sequence according to item [4] or[5], wherein the number of bases in the homologous base sequencedesigned is 13 to 28.

[7] The method for designing the base sequence according to any one ofitems [4] to [6], wherein designing is performed so that at least 80% ofbases in the homologous base sequence designed corresponds to the basesequence searched.

[8] The method for designing the base sequence according to any one ofitems [4] to [7], wherein the 3′ end base of the base sequence searchedis the same as the 3′ end base of the base sequence designed, and the 5′end base of the base sequence searched is the same as the 5′ end base ofthe base sequence designed.

[9] The method for designing the base sequence according to any one ofitems [4] to [8], wherein an overhanging portion is added to the 3′ endof the polynucleotide.

[10] A method for producing a double-stranded polynucleotide comprising:forming one strand by providing an overhanging portion to the 3′ end ofa base sequence homologous to a prescribed sequence which is containedin the base sequences of a target gene and which conforms to thefollowing rules (a) to (d), and forming the other strand by providing anoverhanging portion to the 3′ end of a base sequence complementary tothe base sequence homologous to the prescribed sequence, wherein thenumber of bases in each strand is 15 to 30:

(a) The 3′ end base is adenine, thymine, or uracil,

(b) The 5′ end base is guanine or cytosine,

(c) A 7-base sequence from the 3′ end is rich in one or more types ofbases selected from the group consisting of adenine, thymine, anduracil, and

(d) The number of bases is within a range that allows RNA interferenceto occur without causing cytotoxicity.

[11] A double-stranded polynucleotide synthesized by searching asequence segment having 13 to 28 bases, conforming to the followingrules (a) to (d), from the base sequences of a target gene for RNAinterference, forming one strand by providing an overhanging portion tothe 3′ end of a base sequence homologous to a prescribed sequence whichis contained in the base sequences of the target gene and which conformsto the following rules (a) to (d), and forming the other strand byproviding an overhanging portion to the 3′ end of a base sequencecomplementary to the base sequence homologous to the prescribedsequence, wherein the number of bases in each strand is 15 to 30:

(a) The 3′ end base is adenine, thymine, or uracil,

(b) The 5′ end base is guanine or cytosine,

(c) A 7-base sequence from the 3′ end is rich in one or more types ofbases selected from the group consisting of adenine, thymine, anduracil, and

(d) The number of bases is within a range that allows RNA interferenceto occur without causing cytotoxicity.

[12] A method for inhibiting gene expression comprising the steps ofsearching a sequence segment having 13 to 28 bases, conforming to thefollowing rules (a) to (d), from the base sequences of a target gene forRNA interference, synthesizing a double-stranded polynucleotide suchthat one strand is formed by providing an overhanging portion to the 3′end of a base sequence homologous to a prescribed sequence which iscontained in the base sequences of the target gene and which conforms tothe following rules (a) to (d), the other strand is formed by providingan overhanging portion to the 3′ end of a base sequence complementary tothe base sequence homologous to the prescribed sequence, and the numberof bases in each strand is 15 to 30, and adding the synthesizeddouble-stranded polynucleotide to an expression system of the targetgene of which expression is to be inhibited to inhibit the expression ofthe target gene:

(a) The 3′ end base is adenine, thymine, or uracil,

(b) The 5′ end base is guanine or cytosine,

(c) A 7-base sequence from the 3′ end is rich in one or more types ofbases selected from the group consisting of adenine, thymine, anduracil, and

(d) The number of bases is within a range that allows RNA interferenceto occur without causing cytotoxicity.

[13] A base sequence processing apparatus characterized in that itcomprises partial base sequence creation means for acquiring basesequence information of a target gene for RNA interference and creatingpartial base sequence information corresponding to a sequence segmenthaving a predetermined number of bases in the base sequence information;3′ end base determination means for determining whether the 3′ end basein the partial base sequence information created by the partial basesequence creation means is adenine, thymine, or uracil; 5′ end basedetermination means for determining whether the 5′ end base in thepartial base sequence information created by the partial base sequencecreation means is guanine or cytosine; predetermined base inclusiondetermination means for determining whether base sequence informationcomprising 7 bases at the 3′ end in the partial base sequenceinformation created by the partial base sequence creation means is richin one or more types of bases selected from the group consisting ofadenine, thymine, and uracil; and prescribed sequence selection meansfor selecting prescribed sequence information which specifically causesRNA interference in the target gene from the partial base sequenceinformation created by the partial base sequence creation means, basedon the results determined by the 3′ base determination means, the 5′ endbase determination means, and the predetermined base inclusiondetermination means.

[14] The base sequence processing apparatus according to item [13],characterized in that the partial base sequence creation means furthercomprises region-specific base sequence creation means for creating thepartial base sequence information having the predetermined number ofbases from a segment corresponding to a coding region or transcriptionregion of the target gene in the base sequence information.

[15] The base sequence processing apparatus according to item [13] or[14], characterized in that the partial base sequence creation meansfurther comprises common base sequence creation means for creating thepartial base sequence information having the predetermined number ofbases which is common in a plurality of base sequence informationderived from different organisms.

[16] The base sequence processing apparatus according to any one ofitems [13] to [15], characterized in that the base sequence informationthat is rich corresponds to base sequence information comprising the 7bases containing at least 3 bases which are one or more types of basesselected from the group consisting of adenine, thymine, and uracil.

[17] The base sequence processing apparatus according to any one ofitems [13] to [161, wherein the predetermined number of bases is 13 to28.

[18] The base sequence processing apparatus according to any one ofitems [13] to [17], characterized in that the partial base sequencecreation means further comprises overhanging portion-containing basesequence creation means for creating the partial base sequenceinformation containing an overhanging portion.

[19] The base sequence processing apparatus according to any one ofitems [13] to [17], characterized in that it comprisesoverhanging-portion addition means for adding an overhanging portion toat least one end of the prescribed sequence information.

[20] The base sequence processing apparatus according to item [18] or[19], wherein the number of bases in the overhanging portion is 2.

[21] The base sequence processing apparatus according to any one ofitems [13] to [20], characterized in that it comprises identical/similarbase sequence search means for searching base sequence information,identical or similar to the prescribed sequence information, from otherbase sequence information, and unrelated gene target evaluation meansfor evaluating whether the prescribed sequence information targets genesunrelated to the target gene based on the identical or similar basesequence information searched by the identical/similar base sequencesearch means.

[22] The base sequence processing apparatus according to item [21],characterized in that the unrelated gene target evaluation means furthercomprises total sum calculation means for calculating the total sum ofreciprocals of the values showing the degree of identity or similaritybased on the total amount of base sequence information on the genesunrelated to the target gene in the identical or similar base sequenceinformation searched by the identical/similar base sequence search meansand the values showing the degree of identity or similarity attached tothe base sequence information on the genes unrelated to the target gene,and total sum-based target evaluation means for evaluating whether theprescribed sequence information targets the genes unrelated to thetarget gene based on the total sum calculated by the total sumcalculation means.

[23] A program for running base sequence processing method on acomputer, characterized in that it comprises a partial base sequencecreation step of acquiring base sequence information of a target genefor RNA interference and creating partial base sequence informationcorresponding to a sequence segment having a predetermined number ofbases in the base sequence information; a 3′ end base determination stepof determining whether the 3′ end base in the partial base sequenceinformation created in the partial base sequence creation step isadenine, thymine, or uracil; a 5′ end base determination step ofdetermining whether the 5′ end base in the partial base sequenceinformation created in the partial base sequence creation step isguanine or cytosine; a predetermined base inclusion determination stepof determining whether base sequence information comprising 7 bases atthe 3′ end in the partial base sequence information created in thepartial base sequence creation step is rich in one or more types ofbases selected from the group consisting of adenine, thymine, anduracil; and a prescribed sequence selection step of selecting, based onthe results determined in the 3′ base determination step, the 5′ endbase determination step, and the predetermined base inclusiondetermination step, prescribed sequence information which specificallycauses RNA interference in the target gene from the partial basesequence information created in the partial base sequence creation step.

[24] A computer-readable recording medium characterized in that theprogram according to item [23] is recorded in the medium.

[25] A base sequence processing system which comprises a base sequenceprocessing apparatus which processing base sequence information of atarget gene for RNA interference and a client apparatus, the basesequence processing apparatus and the client apparatus being connectedto each other via a network in a communicable manner, characterized inthat the client apparatus comprises base sequence transmission means fortransmitting a name of the target gene or the base sequence informationto the base sequence processing apparatus, and prescribed sequenceacquisition means for acquiring prescribed sequence information which istransmitted from the base sequence processing apparatus and whichspecifically causes RNA interference in the target gene, and the basesequence processing apparatus comprises partial base sequence creationmeans for acquiring base sequence information corresponding to the nameof the target gene or the base sequence information transmitted from theclient apparatus and creating partial base sequence informationcorresponding to a sequence segment having a predetermined number ofbases in the base sequence information; 3′ end base determination meansfor determining whether the 3′ end base in the partial base sequenceinformation created by the partial base sequence creation means isadenine, thymine, or uracil; 5′ end base determination means fordetermining whether the 5′ end base in the partial base sequenceinformation created by the partial base sequence creation means isguanine or cytosine; predetermined base inclusion determination meansfor determining whether base sequence information comprising 7 bases atthe 3′ end in the partial base sequence information created by thepartial base sequence creation means is rich in one or more types ofbases selected from the group consisting of adenine, thymine, anduracil; prescribed sequence selection means for selecting the prescribedsequence information from the partial base sequence information createdby the partial base sequence creation means, based on the resultsdetermined by the 3′ base determination means, the 5′ end basedetermination means, and the predetermined base inclusion determinationmeans; and prescribed sequence transmission means for transmitting theprescribed sequence information selected by the prescribed sequenceselection means to the client apparatus.

[26] A base sequence processing method characterized in that itcomprises a partial base sequence creation step of acquiring basesequence information of a target gene for RNA interference and creatingpartial base sequence information corresponding to a sequence segmenthaving a predetermined number of bases in the base sequence information;a 3′ end base determination step of determining whether the 3′ end basein the partial base sequence information created in the partial basesequence creation step is adenine, thymine, or uracil; a 5′ end basedetermination step of determining whether the 5′ end base in the partialbase sequence information created in the partial base sequence creationstep is guanine or cytosine; a predetermined base inclusiondetermination step of determining whether base sequence informationcomprising 7 bases at the 3′ end in the partial base sequenceinformation created in the partial base sequence creation step is richin one or more types of bases selected from the group consisting ofadenine, thymine, and uracil; and a prescribed sequence selection stepof selecting, based on the results determined in the 3′ basedetermination step, the 5′ end base determination step, and thepredetermined base inclusion determination step, prescribed sequenceinformation which specifically causes RNA interference in the targetgene from the partial base sequence information created in the partialbase sequence creation step.

[27] The base sequence processing method according to item

[26], characterized in that the partial base sequence creation stepfurther comprises a region-specific base sequence creation step ofcreating the partial base sequence information having the predeterminednumber of bases from a segment corresponding to a coding region ortranscription region of the target gene in the base sequenceinformation.

[28] The base sequence processing method according to item [26] or [27],characterized in that the partial base sequence creation step furthercomprises a common base sequence creation step for creating the partialbase sequence information having the predetermined number of bases whichis common in a plurality of base sequence information derived fromdifferent organisms.

[29] The base sequence processing method according to any one of items[26] to [28], characterized in that the base sequence information thatis rich corresponds to base sequence information comprising the 7 basescontaining at least 3 bases which are one or more types of basesselected from from the group consisting of adenine, thymine, and uracil.

[30] The base sequence processing method according to any one of items[26] to [29], wherein the predetermined number of bases is 13 to 28.

[31] The base sequence processing method according to any one of items[26] to [30], characterized in that the partial base sequence creationstep further comprises an overhanging portion-containing base sequencecreation step of creating the partial base sequence informationcontaining an overhanging portion.

[32] The base sequence processing method according to any one of items[26] to [30], characterized in that it comprises an overhanging-portionaddition step of adding an overhanging portion to at least one end ofthe prescribed sequence information.

[33] The base sequence processing method according to item [31] or [32],wherein the number of bases in the overhanging portion is 2.

[34] The base sequence processing method according to any one of items[26] to [33], characterized in that it comprises an identical/similarbase sequence search step of searching base sequence informationidentical or similar to the prescribed sequence information from otherbase sequence information, and unrelated gene target evaluation step ofevaluating whether the prescribed sequence information targets genesunrelated to the target gene based on the identical or similar basesequence information searched in the identical/similar base sequencesearch step.

[35] The base sequence processing method according to item [34],characterized in that the unrelated gene target evaluation step-furthercomprises a total sum calculation step of calculating the total sum ofreciprocals of the values showing the degree of identity or similaritybased on the total amount of base sequence information on the genesunrelated to the target gene in the identical or similar base sequenceinformation searched in the identical/similar base sequence search stepand the values showing the degree of identity or similarity attached tothe base sequence information on the genes unrelated to the target gene,and a total sum-based target evaluation step of evaluating whether theprescribed sequence information targets the genes unrelated to thetarget gene based on the total sum calculated in the total sumcalculation step.

[36] The program according to item [23], characterized in that thepartial base sequence creation step further comprises a region-specificbase sequence creation step of creating the partial base sequenceinformation having the predetermined number of bases from a segmentcorresponding to a coding region or transcription region of the targetgene in the base sequence information.

[37] The program according to item [23] or [36], characterized in thatthe partial base sequence creation step further comprises a common basesequence creation step of creating the partial base sequence informationhaving the predetermined number of bases which is common in a pluralityof base sequence information derived from different organisms.

[38] The program according to any one of items [23], [36], and [37],characterized in that the base sequence information that is richcorresponds to base sequence information comprising the 7 basescontaining at least 3 bases which are one or more types of basesselected from the group consisting of adenine, thymine, and uracil.

[39] The program according to any one of items [23], [36], [37], and[38], wherein the predetermined number of bases is 13 to 28.

[40] The program according to any one of items [23], [36], [37], [38],and [39], characterized in that the partial base sequence creation stepfurther comprises an overhanging portion-containing base sequencecreation step of creating the partial base sequence informationcontaining an overhanging portion.

[41] The program according to any one of items [23], [36], [37], [38],and [39], characterized in that it comprises an overhanging-portionaddition step of adding an overhanging portion to at least one end ofthe prescribed sequence information.

[42] The program according to item [40] or [41], wherein the number ofbases in the overhanging portion is 2.

[43] The program according to any one of items [23], [36], [37], [38],[39], [40], [41], and [42], characterized in that it comprises anidentical/similar base sequence search step of searching base sequenceinformation identical or similar to the prescribed sequence informationfrom other base sequence information, and an unrelated gene targetevaluation step of evaluating whether the prescribed sequenceinformation targets genes unrelated to the target gene based on theidentical or similar base sequence information searched in theidentical/similar base sequence search step.

[44] The program according to item [43], characterized in that theunrelated gene target evaluation step further comprises a total sumcalculation step of calculating the total sum of reciprocals of thevalues showing the degree of identity or similarity based on the totalamount of base sequence information on the genes unrelated to the targetgene in the identical or similar base sequence information searched inthe identical/similar base sequence search step and the values showingthe degree of identity or similarity attached to the base sequenceinformation on the genes unrelated to the target gene, and a totalsum-based target evaluation step of evaluating whether the prescribedsequence information targets the genes unrelated to the target genebased on the total sum calculated in the total sum calculation step.

[45] A computer-readable recording medium characterized in that theprogram according to any one of items [23] and [36] to [44] is recordedin the medium.

[46] The base sequence processing system according to item [25],characterized in that, in the base sequence processing apparatus, thepartial base sequence creation means further comprises region-specificbase sequence creation means for creating the partial base sequenceinformation having the predetermined number of bases from a segmentcorresponding to a coding region or transcription region of the targetgene in the base sequence information.

[47] The base sequence processing system according to item [25] or [46],characterized in that, in the base sequence processing apparatus, thepartial base sequence creation means further comprises common basesequence creation means for creating the partial base sequenceinformation having the predetermined number of bases which is common ina plurality of base sequence information derived from differentorganisms.

[48] The base sequence processing system according to any one of items[25], [46], and [47], characterized in that, in the base sequenceprocessing apparatus, the base sequence information that is richcorresponds to base sequence information comprising the 7 basescontaining at least 3 bases which are one or more types of basesselected from the group consisting of adenine, thymine, and uracil.

[49] The base sequence processing system according to any one of items[25], [46], [47], and [48], wherein, in the base sequence processingapparatus, the predetermined number of bases is 13 to 28.

[50] The base sequence processing system according to any one of items[25], [46], [47], [48], and [49], characterized in that, in the basesequence processing apparatus, the partial base sequence creation meansfurther comprises overhanging portion-containing base sequence creationmeans for creating the partial base sequence information containing anoverhanging portion.

[51] The base sequence processing system according to any one of items[25], [46], [47], [48], and [49], characterized in that the basesequence processing apparatus comprises overhanging-portion additionmeans for adding an overhanging portion to at least one end of theprescribed sequence information.

[52] The base sequence processing system according to item [50] or [51],wherein, in the base sequence processing apparatus, the number of basesin the overhanging portion is 2.

[53] The base sequence processing system according to any one of items[25], [46], [47], [48], [49], [50], [51], and [52], characterized inthat the base sequence processing apparatus comprises identical/similarbase sequence search means for searching base sequence informationidentical or similar to the prescribed sequence information from otherbase sequence information, and unrelated gene target evaluation meansfor evaluating whether the prescribed sequence information targets genesunrelated to the target gene based on the identical or similar basesequence information searched by the identical/similar base sequencesearch means.

[54] The base sequence processing system according to item [53],characterized in that, in the base sequence processing apparatus, theunrelated gene target evaluation means further comprises total sumcalculation means for calculating the total sum of reciprocals of thevalues showing the degree of identity or similarity based on the totalamount of base sequence information on the genes unrelated to the targetgene in the identical or similar base sequence information searched bythe identical/similar base sequence search means and the values showingthe degree of identity or similarity attached to the base sequenceinformation on the genes unrelated to the target gene, and totalsum-based target evaluation means for evaluating whether the prescribedsequence information targets the genes unrelated to the target genebased on the total sum calculated by the total sum calculation means.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram which shows the designing of siRNA corresponding tosequences common to human and mice.

FIG. 2 is a diagram which shows the regularity of siRNA exhibiting anRNAi effect.

FIG. 3 is a diagram which shows common segments (shown in bold letters)having prescribed sequences in the base sequences of human FBP1 andmouse Fbp1.

FIG. 4 is a diagram listing prescribed sequences common to human FBP1and mouse Fbp1.

FIG. 5 is a diagram in which the prescribed sequences common to humanFBP1 and mouse Fbp1 are scored.

FIG. 6 is a diagram showing the results of BLAST searches on one of theprescribed sequences performed so that genes other than the target arenot knocked out.

FIG. 7 is a diagram showing the results of BLAST searches on one of theprescribed sequences performed so that genes other than the target arenot knocked out.

FIG. 8 is a diagram showing an output result of a program.

FIG. 9 is a diagram which shows the designing of RNA fragments (a to p).

FIG. 10 is a diagram showing the results of testing whether siRNA a to pexhibited an RNAi effect, in which “B” shows the results in drosophilacultured cells, and “C” shows the results in human cultured cells.

FIG. 11 is a diagram showing the analysis results concerning thecharacteristics of sequences of siRNA a to p.

FIG. 12 is a principle diagram showing the basic principle of thepresent invention.

FIG. 13 is a block diagram which shows an example of the configurationof a base sequence processing apparatus 100 of the system to which thepresent invention is applied.

FIG. 14 is a diagram which shows an example of information stored in atarget gene base sequence file 106 a.

FIG. 15 is a diagram which shows an example of information stored in apartial base sequence file 106 b.

FIG. 16 is a diagram which shows an example of information stored in adetermination result file 106 c.

FIG. 17 is a diagram which shows an example of information stored in aprescribed sequence file 106 d.

FIG. 18 is a diagram which shows an example of information stored in areference sequence database 106 e.

FIG. 19 is a diagram which shows an example of information stored in adegree of identity or similarity file 106 f.

FIG. 20 is a diagram which shows an example of information stored in anevaluation result file 106 g.

FIG. 21 is a block diagram which shows an example of the structure of apartial base sequence creation part 102 a of the system to which thepresent invention is applied.

FIG. 22 is a block diagram which shows an example of the structure of anunrelated gene target evaluation part 102 h of the system to which thepresent invention is applied.

FIG. 23 is a flowchart which shows an example of the main processing ofthe system in the embodiment.

FIG. 24 is a flowchart which shows an example of the unrelated geneevaluation process of the system in the embodiment.

FIG. 25 is a diagram which shows the structure of a target expressionvector pTREC.

FIG. 26 is a diagram which shows the results of PCR in which one of theprimers in Example 2, 2. (2) is designed such that no intron isinserted.

FIG. 27 is a diagram which shows the results of PCR in which one of theprimers in Example 2, 2. (2) is designed such that an intron isinserted.

FIG. 28 is a diagram which shows the sequence and structure of siRNA;siVIM35.

FIG. 29 is a diagram which shows the sequence and structure of siRNA;siVIM812.

FIG. 30 is a diagram which shows the sequence and structure of siRNA;siControl.

FIG. 31 is a diagram which shows the results of assay of RNAi activityof siVIM812 and siVIM35.

FIG. 32 is a diagram which shows RNAi activity of siControl, siVIM812,and siVIM35 against vimentin.

FIG. 33 is a diagram which shows the results of antibody staining.

FIG. 34 is a diagram which shows the assay results of RNAi activity ofsiRNA designed by the program against the luciferase gene.

FIG. 35 is a diagram which shows the assay results of RNAi activity ofsiRNA designed by the program against the sequences of SARS virus.

BEST MODE FOR CARRYING OUT THE INVENTION

The embodiments of the present invention will be described below in theorder of the columns <1> to <7>.

<1> Method for searching target base sequence of RNA interference

<2> Method for designing base sequence of polynucleotide for causing RNAinterference

<3> Method for producing double-stranded polynucleotide

<4> Method for inhibiting gene expression

<5> siRNA sequence design program

<6> siRNA sequence design business model system

<7> Base sequence processing apparatus for running siRNA sequence designprogram, etc.

<1> Method for Searching Target Base Sequence of RNA Interference

The search method of the present invention is a method for searching abase sequence, which causes RNA interference, from the base sequences ofa target gene. Specifically, in the search method of the presentinvention, a sequence segment conforming to the following rules (a) to(d) is searched from the base sequences of a target gene for RNAinterference.

(a) The 3′ end base is adenine, thymine, or uracil.

(b) The 5′ end base is guanine or cytosine.

(c) A 7-base sequence from the 3′ end is rich in one or more types ofbases selected from the group consisting of adenine, thymine, anduracil.

(d) The number of bases is within a range that allows RNA interferenceto occur without causing cytotoxicity.

The term “gene” in the term “target gene” means a medium which codes forgenetic information. The “gene” consists of a substance, such as DNA,RNA, or a complex of DNA and RNA, which codes for genetic information.As the genetic information, instead of the substance itself, electronicdata of base sequences can be handled in a computer or the like. The“target gene” may be set as one coding region, a plurality of codingregions, or all the polynucleotides whose sequences have been revealed.When a gene with a particular function is desired to be searched, bysetting only the particular gene as the target, it is possible toefficiently search the base sequences which cause RNA interferencespecifically in the particular gene. Namely, RNA interference is knownas a phenomenon which destructs mRNA by interference, and by selecting aparticular coding region, search load can be reduced. Moreover, a groupof transcription regions may be treated as the target region to besearched. Additionally, in the present specification, base sequences areshown on the basis of sense strands, i.e., sequences of mRNA, unlessotherwise described. Furthermore, in the present specification, a basesequence which satisfies the rules (a) to (d) is referred to as a“prescribed sequence”. In the rules, thymine corresponds to a DNA basesequence, and uracil corresponds to an RNA base sequence.

The rule (c) regulates so that a sequence in the vicinity of the 3′ endcontains a rich amount of type(s) of base(s) selected from the groupconsisting of adenine, thymine, and uracil, and more specifically, as anindex for search, regulates so that a 7-base sequence from the 3′ end isrich in one or more types of bases selected from adenine, thymine, anduracil.

In the rule (c), the phrase “sequence rich in” means that the frequencyof a given base appearing is high, and schematically, a 5 to 10-basesequence, preferably a 7-base sequence, from the 3′ end in theprescribed sequence contains one or more types of bases selected fromadenine, thymine, and uracil in an amount of preferably at least 40% ormore, and more preferably at least 50%. More specifically, for example,in a prescribed sequence of about 19 bases, among 7 bases from the 3′end, preferably at least 3 bases, more preferably at least 4 bases, andparticularly preferably at least 5 bases, are one or more types of basesselected from the group consisting of adenine, thymine, and uracil.

The means for confirming the correspondence to the rule (c) is notparticularly limited as long as it can be confirmed that preferably atleast 3 bases, more preferably at least 4 bases, and particularlypreferably at least 5 bases, among 7 bases are adenine, thymine, oruracil. For example, a case, wherein inclusion of 3 or more bases whichcorrespond to one or more types of bases selected from the groupconsisting of adenine, thymine, and uracil in a 7-base sequence from the3′ end is defined as being rich, will be described below. Whether thebase is any one of the three types of bases is checked from the firstbase at the 3′ end one after another, and when three corresponding basesappear by the seventh base, conformation to the rule (c) is determined.For example, if three corresponding bases appear by the third base,checking of three bases is sufficient. That is, in the search withrespect to the rule (c), it is not always necessary to check all of theseven bases at the 3′ end. Conversely, non-appearance of three or morecorresponding bases by the seventh base means being not rich, thus beingdetermined that the rule (c) is not satisfied.

In a double-stranded polynucleotide, it is well-known that adeninecomplementarily forms hydrogen-bonds to thymine or uracil. In thecomplementary hydrogen bond between guanine and cytosine (G-C hydrogenbond), three hydrogen bonding sites are formed. On the other hand, thecomplementary hydrogen bond between adenine and thymine or uracil(A-(T/U) hydrogen bond) includes two hydrogen bonding sites. Generallyspeaking, the bonding strength of the A-(T/U) hydrogen bond is weakerthan that of the G-C hydrogen bond.

In the rule (d), the number of bases of the base sequence to be searchedis regulated. The number of bases of the base sequence to be searchedcorresponds to the number of bases capable of causing RNA interference.Depending on the conditions, for example the species of an organism, incases of siRNA having an excessively large number of bases, cytotoxicityis known to occur. The upper limit of the number of bases variesdepending on the species of organism to which RNA interference isdesired to be caused. The number of bases of the single strandconstituting siRNA is preferably 30 or less regardless of the species.Furthermore, in mammals, the number of bases is preferably 24 or less,and more preferably 22 or less. The lower limit, which is notparticularly limited as long as RNA interference is caused, ispreferably at least 15, more preferably at least 18, and still morepreferably at least 20. With respect to the number of bases as a singlestrand constituting siRNA, searching with a number of 21 is particularlypreferable.

Furthermore, although a description will be made below, in siRNA, anoverhanging portion is provided at the 3′ end of the prescribedsequence. The number of bases in the overhanging portion is preferably2. Consequently, the upper limit of the number of bases in theprescribed sequence only, excluding the overhanging portion, ispreferably 28 or less, more preferably 22 or less, and still morepreferably 20 or less, and the lower limit is preferably at least 13,more preferably at least 16, and still more preferably at least 18. Inthe prescribed sequence, the most preferable number of bases is 19. Thetarget base sequence for RNAi may be searched either including orexcluding the overhanging portion.

Base sequences conforming to the prescribed sequence have an extremelyhigh probability of causing RNA interference. Consequently, inaccordance with the search method of the present invention, it ispossible to search sequences that cause RNA interference with extremelyhigh probability, and designing of polynucleotides which cause RNAinterference can be simplified.

In another preferred example, the prescribed sequence does not contain asequence in which 7 or more bases of guanine (G) and/or cytosine (C) arecontinuously present. Examples of the sequence in which 7 or more basesof guanine and/or cytosine are continuously present include a sequencein which either guanine or cytosine is continuously present as well as asequence in which a mixed sequence of guanine and cytosine is present.More specific examples include GGGGGGG, CCCCCCC, and a mixed sequence ofGCGGCCC.

Furthermore, in the search of the prescribed sequence, detection can beefficiently performed by using a computer installed with a program whichallows a search of segments conforming to the rules (a) to (c), etc.,after determining the number of bases. More specific embodiments will bedescribed below in the columns <5> siRNA sequence design program and <7>Base sequence processing apparatus for running siRNA sequence designprogram.

<2> Method for Designing Base Sequence of Polynucleotide for Causing RNAInterference

In the method for designing a base sequence in accordance with thepresent invention, a base sequence of polynucleotide which causes RNAinterference (siRNA) is designed on the basis of the base sequencesearched by the search method described above. siRNA is mainly composedof RNA. siRNA which partially contains DNA, i.e., a hybridpolynucleotide, is also included in the examples of siRNA. In the methodfor designing a base sequence in accordance with the present invention,a base sequence conforming to the rules (a) to (d) is searched from thebase sequences of a target gene, and a base sequence homologous to thesearched base sequence is designed. In another preferred design example,it may be possible to take into consideration a case in which theprescribed sequence does not contain a sequence in which 7 or more basesof guanine (G) and/or cytosine (C) are continuously present. The rules(a) to (d) and the search method are the same as those described aboveregarding the search method of the present invention.

The term “homologous sequence” refers to the same sequence and asequence in which mutations, such as deletions, substitutions, andadditions, have occurred to the same sequence to an extent that thefunction of causing the RNA interference has not been lost. Althoughdepending on the conditions, such as the type and sequence of the targetgene, the range of the allowable mutation, in terms of homology, ispreferably 80% or more, more preferably 90% or more, and still morepreferably 95% or more. When homology in the range of the allowablemutation is calculated, desirably, the numerical values calculated usingthe same search algorithm are compared. The search algorithm is notparticularly limited. A search algorithm suitable for searching forlocal sequences is preferable. More specifically, BLAST, ssearch, or thelike is preferably used.

As described above, although slight modification of the searchedsequence is allowable, it is particularly preferred that the number ofbases in the base sequence to be designed be the same as that of thesearched sequence. For example, with respect to the allowance for changeunder the same number of bases, the bases of the base sequence to bedesigned correspond to those of the sequence searched at a rate ofpreferably 80% or more, more preferably 90% or more, and particularlypreferably 95% or more. For example, when a base sequence having 19bases is designed, preferably 16 or more bases, more preferably 18 ormore bases, correspond to those of the searched base sequence.Furthermore, when a sequence homologous to the searched base sequence isdesigned, desirably, the 3′ end base of the base sequence searched isthe same as the 3′ end base of the base sequence designed, and alsodesirably, the 5′ end base of the base sequence searched is the same asthe 5′ end base of the base sequenced designed.

An overhanging portion is usually provided on a siRNA molecule. Theoverhanging portion is a protrusion provided on the 3′ end of eachstrand in a double-stranded RNA molecule. Although depending on thespecies of organism, the number of bases in the overhanging portion ispreferably 2. Basically, any base sequence is acceptable in theoverhanging portion. In some cases, the same base sequence as that ofthe target gene to be searched, TT, UU, or the like may be preferablyused. As described above, by providing the overhanging portion at the 3′end of the prescribed sequence which has been designed so as to behomologous to the base sequence searched, a sense strand constitutingsiRNA is designed.

Alternatively, it may be possible to search the prescribed sequence withthe overhanging portion being included from the start to performdesigning. The preferred number of bases in the overhanging portion is2. Consequently, for example, in order to design a single strandconstituting siRNA including a prescribed sequence having 19 bases andan overhanging portion having 2 bases, as the number of bases of siRNAincluding the overhanging portion, a sequence of 21 bases is searchedfrom the target gene. Furthermore, when a double-stranded state issearched, a sequence of 23 bases may be searched.

In the method for designing a base sequence in accordance with thepresent invention, as described above, a given sequence is searched froma desired target gene. The target to which RNA interference is intendedto be caused does not necessarily correspond to the origin of the targetgene, and is also applicable to an analogous species, etc. For example,it is possible to design siRNA used for a second species that isanalogous to a first species using a gene isolated from the firstspecies as a target gene. Furthermore, it is possible to design siRNAthat can be widely applied to mammals, for example, by searching acommon sequence from two or more species of mammals and searching aprescribed sequence from the common sequence to perform designing. Thereason for this is that it is highly probable that the sequence commonto two or more mammals exists in other mammals.

In order to prevent RNA interference from occurring in genes not relatedto the target gene, preferably, a search is made to determine whether asequence that is identical or similar to the designed sequence isincluded in the other genes. A search for the sequence that is identicalor similar to the designed sequence may be performed using softwarecapable of performing a general homology search, etc. By excluding suchan identical/similar sequence, it is possible to design a sequence whichcauses RNA interference specifically to the target gene only.

In the design method of the present invention, RNA molecules that causeRNA interference can be easily designed with high probability. Althoughsynthesis of RNA still requires effort, time, and cost, the designmethod of the present invention can greatly minimize them.

<3> Method for Producing Double-Stranded Polynucleotide

By the method for producing a double-stranded polynucleotide inaccordance with the present invention, a double-stranded polynucleotidethat has a high probability of causing RNA interference can be produced.For the double-stranded polynucleotide of the present invention, a basesequence of the polynucleotide is designed in accordance with the methodfor designing the base sequence of the present invention describedabove, and a double-stranded polynucleotide is synthesized so as tofollow the sequence design. Preferred embodiments in the sequence designare the same as those described above regarding the method for designingthe base sequence.

The double-stranded polynucleotide synthesized causes RNA interference,and siRNA is known as such a double-stranded polynucleotide.Additionally, the double-stranded polynucleotide produced by theproduction method of the present invention is preferably composed ofRNA, but a hybrid polynucleotide which partially includes DNA may beacceptable. In this specification, double-stranded polynucleotidespartially including DNA are also contained in the concept of siRNA.According to the research conducted by the present inventors, siRNAtends to have structural and functional asymmetry, and in view of theobject of causing RNA interference, a half of the sense strand at the 5′end side and a half of the antisense strand at the 3′ end side aredesirably composed of RNA.

In a double-stranded polynucleotide, one strand is formed by providingan overhanging portion to the 3′ end of a base sequence homologous tothe prescribed sequence conforming to the rules (a) to (d) contained inthe base sequence of the target gene, and the other strand is formed byproviding an overhanging portion to the 3′ end of a base sequencecomplementary to the base sequence homologous to the prescribedsequence. The number of bases in each strand, including the overhangingportion, is 18 to 24, more preferably 20 to 22, and particularlypreferably 21. The number of bases in the overhanging portion ispreferably 2. siRNA having 21 bases in total in which the overhangingportion is composed of 2 bases is suitable for causing RNA interferencewith high probability without causing cytotoxicity even in mammals.

RNA may be synthesized, for example, by chemical synthesis or bystandard biotechnology. In one technique, a DNA strand having apredetermined sequence is produced, single-stranded RNA is synthesizedusing the produced DNA strand as a template in the presence of atranscriptase, and the synthesized single-stranded RNA is formed intodouble-stranded RNA.

With respect to the basic technique for molecular biology, there aremany standard, experimental manuals, for example, BASIC METHODS INMOLECULAR BIOLOGY (1986); Sambrook et al., MOLECULAR CLONING; ALABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989); Saibo-Kogaku Handbook (Handbook forcell engineering), edited by Toshio Kuroki et al., Yodosha (1992); andShin-Idenshi-Kogaku Handbook (New handbook for genetic engineering),edited by Muramatsu et al., Yodosha (1999).

One preferred embodiment of polynucleotide produced by the productionmethod of the present invention is a double-stranded polynucleotideproduced by a method in which a sequence segment including 13 to 28bases conforming to the rules (a) to (d) is searched from a basesequence of a target gene for RNA interference, one strand is formed byproviding an overhanging portion at the 3′ end of a base sequencehomologous to the prescribed sequence following the rules (a) to (d),the other strand is formed by providing an overhanging portion at the 3′end of a sequence complementary to the base sequence homologous to theprescribed sequence, and synthesis is performed so that the number ofbases in each strand is 15 to 30. The resulting polynucleotide has ahigh probability of causing RNA interference.

It is also possible to prepare an expression vector which expressessiRNA. By placing a vector which expresses a sequence containing theprescribed sequence under a condition of a cell line or cell-free systemin which expression is allowed to occur, it is possible to supplypredetermined siRNA using the expression vector.

Since conventional designing of siRNA has depended on the experiencesand intuition of the researcher, trial and error have often beenrepeated. However, by the double-stranded polynucleotide productionmethod in accordance with the present invention, it is possible toproduce a double-stranded polynucleotide which causes RNA interferencewith high probability. In accordance with the search method, sequencedesign method, or polynucleotide production method of the presentinvention, it is possible to greatly reduce effort, time, and costrequired for various experiments, manufacturing, etc., which use RNAinterference. Namely, the present invention greatly simplifies variousexperiments, research, development, manufacturing, etc., in which RNAinterference is used, such as gene analysis, search for targets for newdrug development, development of new drugs, gene therapy, and researchon differences between species, and thus efficiency can be improved.

<4> Method for Inhibiting Gene Expression

The method for inhibiting gene expression in accordance with the presentinvention includes a step of searching a predetermined base sequence, astep of designing and synthesizing a base sequence of siRNA based on thesearched base sequence, and a step of introducing the resulting siRNAinto an expression system containing a target gene.

The step of searching the predetermined base sequence follows the methodfor searching the target base sequence for RNA interference describedabove. Preferred embodiments are the same as those described above. Thestep of designing and synthesizing the base sequence of siRNA based onthe searched base sequence can be carried out in accordance with themethod for designing the base sequence of the polynucleotide for causingRNA interference and the method for producing the double-strandedpolynucleotide described above. Preferred embodiments are the same asthose described above.

The resulting double-stranded polynucleotide is added to an expressionsystem for a target gene to inhibit the expression of the target gene.The expression system for a target gene means a system in which thetarget gene is expressed, and more specifically, a system provided witha reaction system in which at least mRNA of the target gene is formed.Examples of the expression system for the target gene include both invitro and in vivo systems. In addition to cultured cells, culturedtissues, and living bodies, cell-free systems can also be used as theexpression system for the target genes. The target gene of whichexpression is intended to be inhibited (inhibition target gene) is notnecessarily a gene of a species corresponding to the origin of thesearched sequence. However, as the relationship between the origin ofthe search target gene and the origin of the inhibition target genebecomes closer, a predetermined gene can be more specifically andeffectively inhibited.

Introduction into an expression system means incorporation into theexpression reaction system for the target gene. For example, in onemethod, a double-stranded nucleotide is transfected to a cultured cellincluding a target gene and incorporated into the cell. In anothermethod, an expression vector having a base sequence comprising aprescribed sequence and an overhanging portion is formed, and theexpression vector is introduced into a cell having a target gene.

In accordance with the gene inhibition method of the present invention,since polynucleotides which cause RNA interference can be efficientlyproduced, it is possible to inhibit genes efficiently and simply.

<5> siRNA Sequence Design Program

Embodiments of the siRNA sequence design program will be describedbelow.

(5-1) Outline of the Program

When species whose genomes are not sequenced, for example, horse andswine, are subjected to RNA interference, this program calculates asequence of siRNA usable in the target species based on publishedsequence information regarding human beings and mice. If siRNA isdesigned using this program, RNA interference can be carried out rapidlywithout sequencing the target gene. In the design (calculation) ofsiRNA, sequences having RNAi activity with high probability are selectedin consideration of the rules of allocation of G or C (the rules (a) to(d) described above), and checking is performed by homology search sothat RNA interference does not occur in genes that are not related tothe target gene. In this specification, “G or C” may also be written as“G/C”, and “A or T” may also be written as “A/T”. Furthermore, “T(U)” in“A/T(U)” means T (thymine) in the case of sequences of deoxyribonucleicacid and U (uracil) in the case of sequences of ribonucleic acid.

(5-2) Policy of siRNA Design

Sequences of human gene X and mouse gene X which are homologous to thehuman gene are assumed to be known. This program reads the sequences andsearches completely common sequences each having 23 or more bases fromthe coding regions (CDS). By designing siRNA from the common portions,the resulting siRNA can target both human and mouse gene X (FIG. 1).

Since the portions completely common to human beings and mice arebelieved to also exist in other mammals with high probability, the siRNAis expected to act not only on gene X of human beings and mice but alsoon gene X of other mammals. Namely, even if in an animal species inwhich the sequence of a target gene is not known, if sequenceinformation is known regarding the corresponding homologues of humanbeings and mice, it is possible to design siRNA using this program.

Furthermore, in mammals, it is known that sequences of effective siRNAhave regularity (FIG. 2). In this program, only sequences conforming tothe rules are selected. FIG. 2 is a diagram which shows regularity ofsiRNA sequences exhibiting an RNAi effect (rules of G/C allocation ofsiRNA). In FIG. 2, with respect to siRNA in which two RNA strands, eachhaving a length of 21 bases and having an overhang of 2 bases on the 3′side, form base pairs between 19 bases at the 5′ side of the twostrands, the sequence in the coding side among the 19 bases forming thebase pairs must satisfy the following conditions: 1) The 3′ end is A/U;2) the 5′ end is G/C, and 3) 7 characters on the 3′ side has a highratio of A/U. In particular, the conditions 1) and 2) are important.

(5-3) Structure of Program

This program consists of three parts, i.e., (5-3-1) a part whichsearches sequences of sites common to human beings and mice (partialsequences), (5-3-2) a part which scores the sequences according to therules of G/C allocation, and (5-3-3) a part which performs checking byhomology search so that unrelated genes are not targeted.

(5-3-1) Part Which Searches Common Sequences

This part reads a plurality of base sequence files (file 1, file 2, file3, . . . ) and finds all sequences of 23 characters that commonly appearin all the files.

Calculation Example

As file 1, sequences of human gene FBP1 (HM_(—)000507: Homo sapiensfructose-1,6-bisphosphatase 1) and, as file 2, sequences of mouse geneFbp1 (NM_(—)019395: Mus musculus fructose bisphosphatase 1) wereinputted into the program. As a result, from the sequences of the two(FIG. 3), 15 sequences, each having 23 characters, that were common tothe two (sequences common to human FBP1 and mouse Fbp1) were found (FIG.4).

(5-3-2) Part Which Scores Sequences

This part scores the sequences each having 23 characters in order toonly select the sequences conforming to the rules of G/C allocation.

(Method)

The sequences each having 23 characters are scored in the followingmanner.

Score 1: Is the 21st character from the head A/U?

-   -   [no =0, yes=1]

Score 2: Is the third character from the head G/C?

-   -   [no =0, yes=1]

Score 3: The number of A/U among 7 characters between the 15th characterand 21st character from the head

-   -   [0 to 7]

Total score: Product of scores 1 to 3. However, if the product is 3 orless, the total score is considered as zero.

Calculation Example

With respect to 15 sequences in FIG. 4, the results of calculation areshown in FIG. 5. FIG. 5 is a diagram in which the sequences common tohuman FBP1 and mouse Fbp1 are scored. Furthermore, score 1, score 2,score 3, and total score are described in this order after the sequencesshown in FIG. 5.

(5-3-3) Part Which Performs Checking so That Unrelated Genes are NotTargeted

In order to prevent the designed siRNA from acting on genes unrelated tothe target gene, homology search is performed against all the publishedmRNA of human beings and mice, and the degree of unrelated genes beinghit is evaluated. Various search algorithms can be used in the homologysearch. Herein, an example in which BLAST is used will be described.Additionally, when BLAST is used, in view that the sequences to besearched are as short as 23 bases, it is desirable that Word Size bedecreased sufficiently.

After the Blast search, among the hits with an E-value of 10.0 or less,with respect to all the hits other than the target gene, the total sumof the reciprocals of the E-values are calculated (hereinafter, thevalue is referred to as a homology score). Namely, the homology score(X) is found in accordance with the following expression.$X = {\sum\limits_{{all}\quad{hits}}\frac{1}{E}}$

Note: A lower E value of the hit indicates higher homology to 23characters of the query and higher risk of being targeted by siRNA. Alarger number of hits indicates a higher probability that more unrelatedgenes are targeted. In consideration of these two respects, the riskthat siRNA targets genes unrelated to the target gene is evaluated usingthe above expression.

Calculation Example

The results of homology search against the sequences each having 23characters and the homology scores are shown (FIGS. 6 and 7). FIG. 6shows the results of BLAST searches of a sequence common to human FBP1and mouse Fbp1, i.e.,. “caccctgacccgcttcgtcatgg”, and the first twolines are the results in which both mouse Fbp1 and human FBP1 are hit.The homology score is 5.9, and this is an example of a small number ofhits. The risk that siRNA of this sequence targets the other genes islow. Furthermore, FIG. 7 shows the results of BLAST searches of asequence common to human FBP1 and mouse Fbp1, i.e.,“gccttctgagaaggatgctctgc”. This is an example of a large number of hits,and the homology score is 170.8. Since the risk of targeting other genesis high, the sequence is not suitable as siRNA.

In practice, the parts (5-3-1), (5-3-2), and (5-3-3) may be integrated,and when the sequences of human beings and mice shown in FIG. 3 areinputted, an output as shown in FIG. 8 is directly obtained. Herein,after the sequences shown in FIG. 8, score 1, score 2, score 3, totalscore, and the tenfold value of homology score are described in thisorder. Additionally, in order to save processing time, the program maybe designed so that the homology score is not calculated when the totalscore is zero. As a result, it is evident that the segment “36caccctgacccgcttcgtcatgg” can be used as siRNA. Furthermore, one of theparts (5-3-1), (5-3-2), and (5-3-3) may be used independently.

(5-4) Actual Calculation

With respect to about 6,400 gene pairs among the homologues betweenhuman beings and mice, siRNA was actually designed using this program.As a result, regarding about 70% thereof, it was possible to designsiRNA which had a sequence common to human beings and mice and whichsatisfied the rules of effective siRNA sequence regularity so thatunrelated genes were not targeted.

These siRNA sequences are expected to effectively inhibit target genesnot only in human beings and mice but also in a wide range of mammals,and are believed to have a high industrial value, such as applicationsto livestock and pet animals. Moreover, it is possible to design siRNAwhich simultaneously targets two or more genes of the same species,e.g., eIF2Cl and eIF2C2, using this program. Thus, the method fordesigning siRNA provided by this program has a wide range of applicationand is extremely strong. In further application, by designing a PCRprimer using a sequence segment common to human beings and mice, targetgenes can be amplified in a wide range of mammals.

Additionally, embodiments of the apparatus which runs the siRNA sequencedesign program will be described in detail below in the column <7> Basesequence processing apparatus for running siRNA sequence design program.

<6> siRNA Sequence Design Business Model System

In the siRNA sequence design business model system of the presentinvention, when the siRNA sequence design program is applied, the systemrefers to a genome database, an EST database, and a phylogenetic treedatabase, alone or in combination, according to the logic of thisprogram, and effective siRNA in response to availability of genesequence information is proposed to the client. The term “availability”means a state in which information is available.

(1) In a case in which it is difficult to specify an ORF although genomeinformation is available, siRNA candidates effective against assumedexon sites are extracted based on EST information, etc., and siRNAsequences in consideration of splicing variants and evaluation resultsthereof are displayed.

(2) In a case in which a gene sequence and a gene name are known, afterthe input of the gene sequence or the gene name, effective siRNAcandidates are extracted, and siRNA sequences and evaluation resultsthereof are displayed.

(3) In a case in which genome information is not available, using thegene sequences of a related species storing the same type of genefunctions (congeneric or having the same origin) or gene sequences oftwo or more species which have a short distance in phylogenetic treesand of which genome sequences are available, effective siRNA candidatesare extracted, and siRNA sequences and evaluation results thereof aredisplayed.

(4) In order to analyze functions of genes relating infectious diseasesand search for targets for new drug development, a technique iseffective in which the genome database and phylogenetic tree database ofmicroorganisms are further combined with apoptosis induction siteinformation and function expression site information of microorganismsto obtain exhaustive siRNA candidate sequences.

<7> Base Sequence Processing Apparatus for Running siRNA Sequence DesignProgram, etc.

Embodiments of the base sequence processing apparatus which is anapparatus for running the siRNA sequence design program described above,the program for running a base sequence processing method on a computer,the recording medium, and the base sequence processing system inaccordance with the present invention will be described in detail belowwith reference to the drawings. However, it is to be understood that thepresent invention is not restricted by the embodiments.

SUMMARY OF THE PRESENT INVENTION

The summary of the present invention will be described below, and thenthe constitution, processing, etc., of the present invention will bedescribed in detail. FIG. 12 is a principle diagram showing the basicprinciple of the present invention.

Overall, the present invention has the following basic features. Thatis, in the present invention, base sequence information of a target genefor RNA interference is obtained, and partial base sequence informationcorresponding to a sequence segment having a predetermined number ofbases in the base sequence information is created (step S-1).

In step S-1, partial base sequence information having a predeterminednumber of bases may be created from a segment corresponding to a codingregion or transcription region of the target gene in the base sequenceinformation. Furthermore, partial base sequence information having apredetermined number of bases which is common in a plurality of basesequence information derived from different organisms (e.g., human basesequence information and mouse base sequence information) may becreated. Furthermore, partial base sequence information having apredetermined number of bases which is common in a plurality ofanalogous base sequence information in the same species may be created.Furthermore, common partial base sequence information having apredetermined number of bases may be created from segments correspondingto coding regions or transcription regions of the target gene in aplurality of base sequence information derived from different species.Furthermore, common partial base sequence information having apredetermined number of bases may be created from segments correspondingto coding regions or transcription regions of the target gene in aplurality of analogous base sequence information in the same species.Consequently, a prescribed sequence which specifically causes RNAinterference in the target gene can be efficiently selected, andcalculation load can be reduced.

Furthermore, in step S-1, partial base sequence information including anoverhanging portion may be created. Specifically, for example, partialbase sequence information to which overhanging portion inclusioninformation, which shows that an overhanging portion is included, isadded may be created. Namely, partial base sequence information andoverhanging portion inclusion information may be correlated with eachother. Thereby, it becomes possible to select the prescribed sequencewith the overhanging portion being included from the start to performdesigning.

The upper limit of the predetermined number of bases is, in the case ofnot including the overhanging portion, preferably 28 or less, morepreferably 22 or less, and still more preferably 20 or less, and in thecase of including the overhanging portion, preferably 32 or less, morepreferably 26 or less, and still more preferably 24 or less. The lowerlimit of the predetermined number of bases is, in the case of notincluding the overhanging portion, preferably at least 13, morepreferably at least 16, and still more preferably at least 18, and inthe case of including the overhanging portion, preferably at least 17,more preferably at least 20, and still more preferably at least 22. Mostpreferably, the predetermined number of bases is, in the case of notincluding the overhanging portion, 19, and in the case of including theoverhanging portion, 23. Thereby, it is possible to efficiently selectthe prescribed sequence which causes RNA interference without causingcytotoxicity even in mammals.

Subsequently, it is determined whether the 3′ end base in the partialbase sequence information created in step S-1 is adenine, thymine, oruracil (step S-2). Specifically, for example, when the 3′ end base isadenine, thymine, or uracil, “1” may be outputted as the determinationresult, and when it is not, “0” may be outputted.

Subsequently, it is determined whether the 5′ end base in the partialbase sequence information created in step S-1 is guanine or cytosine(step S-3). Specifically, for example, when the 5′ end base is guanineor cytosine, “1” may be outputted as the determination result, and whenit is not, “0” may be outputted.

Subsequently, it is determined whether base sequence informationcomprising 7 bases at the 3′ end in the partial base sequenceinformation created in step S-1 is rich in one or more types of basesselected from the group consisting of adenine, thymine, and uracil (stepS-4). Specifically, for example, the number of bases of one or moretypes of bases selected from the group consisting of adenine, thymine,and uracil contained in the base sequence information comprising 7 basesat the 3′ end may be outputted as the determination result. The rule ofdetermination in step S-4 regulates that base sequence information inthe vicinity of the 3′ end of the partial base sequence informationcreated in step S-1 contains a rich amount of one or more types of basesselected from the group consisting of adenine, thymine, and uracil, andmore specifically, as an index for search, regulates that the basesequence information in the range from the 3′ end base to the seventhbase from the 3′ end is rich in one or more types of bases selected fromthe group consisting of adenine, thymine, and uracil.

In step S-4, the phrase “base sequence information rich in” correspondsto the phrase “sequence rich in” described in the column <1> Method forsearching target base sequence for RNA interference. Specifically, forexample, when the partial base sequence information created in step S-1comprises about 19 bases, in the base sequence information comprising 7bases in the partial base sequence information, preferably at least 3bases, more preferably at least 4 bases, and particularly preferably atleast 5 bases, are one or more types of bases selected from the groupconsisting of adenine, thymine, and uracil.

Furthermore, in steps S-2 to S-4, when partial base sequence informationincluding the overhanging portion is determined, the sequence segmentexcluding the overhanging portion in the partial base sequenceinformation is considered as the determination target.

Subsequently, based on the determination results in steps S-2, S-3, andS-4, prescribed sequence information which specifically causes RNAinterference in the target gene is selected from the partial basesequence information created in step S-1 (Step S-5).

Specifically, for example, partial base sequence information in whichthe 3′ end base has been determined as adenine, thymine, or uracil instep S-2, the 5′ end base has been determined as guanine or cytosine instep S-3, and base sequence information comprising 7 bases at the 3′ endin the partial base sequence information has been determined as beingrich in one or more types of bases selected from the group consisting ofadenine, thymine, and uracil is selected as prescribed sequenceinformation. Specifically, for example, a product of the valuesoutputted in steps S-2, S-3, and S-4 may be calculated, and based on theproduct, prescribed sequence information may be selected from thepartial base sequence information created in step S-1.

Consequently, it is possible to efficiently and easily produce a siRNAsequence which has an extremely high probability of causing RNAinterference, i.e., which is effective for RNA interference, in mammals,etc.

Here, an overhanging portion may be added to at least one end of theprescribed sequence information selected in-step S-5. Additionally, forexample, when a target is searched, the overhanging portion may be addedto both ends of the prescribed sequence information. Consequently,designing of a polynucleotide which causes RNA interference can besimplified.

Additionally, the number of bases in the overhanging portion correspondsto the number of bases described in the column <2> Method for designingbase sequence of polynucleotide for causing RNA interference.Specifically, for example, 2 is particularly suitable as the number ofbases.

Furthermore, base sequence information that is identical or similar tothe prescribed sequence information selected in step S-5 may be searchedfrom other base sequence information (e.g., base sequence informationpublished in a public database, such as RefSeq (Reference Sequenceproject) of NCBI) using a known homology search method, such as BLAST,FASTA, or ssearch, and based on the searched identical or similar basesequence information, evaluation may be made whether the prescribedsequence information targets genes unrelated to the target gene.

Specifically, for example, base sequence information that is identicalor similar to the prescribed sequence information selected in step S-5is searched from other base sequence information (e.g., base sequenceinformation published in a public database, such as RefSeq of NCBI)using a known homology search method, such as BLAST, FASTA, or ssearch.Based on the total amount of base sequence information on the genesunrelated to the target gene in the searched identical or similar basesequence information and the values showing the degree of identity orsimilarity (e.g., “E value” in BLAST, FASTA, or ssearch) attached to thebase sequence information on the genes unrelated to the target gene, thetotal sum of the reciprocals of the values showing the degree ofidentity or similarity is calculated, and based on the calculated totalsum (e.g., based on the size of the total sum calculated), evaluationmay be made whether the prescribed sequence information targets genesunrelated to the target gene.

Consequently, it is possible to select a sequence which specificallycauses RNA interference only to the target gene.

If RNA is synthesized based on the prescribed sequence information whichis selected in accordance with the present invention and which does notcause RNA interference in genes unrelated to the target gene, it ispossible to greatly reduce effort, time, and cost required compared withconventional techniques.

[System Configuration]

First, the configuration of this system will be described. FIG. 13 is ablock diagram which shows an example of the system to which the presentinvention is applied and which conceptually shows only the parts relatedto the present invention.

Schematically, in this system, a base sequence processing apparatus 10which processes base sequence information of a target gene for RNAinterference and an external system 200 which provides externaldatabases regarding sequence information, structural information, etc.,and external programs, such as homology search, are connected to eachother via a network 300 in a communicable manner.

In FIG. 13, the network 300 has a function of interconnecting betweenthe base sequence processing apparatus 100 and the external system 200,and is, for example, the Internet.

In FIG. 13, the external system 200 is connected to the base sequenceprocessing apparatus 100 via the network 300, and has a function ofproviding the user with the external databases regarding sequenceinformation, structural information, etc., and Web sites which executeexternal programs, such as homology search and motif search.

The external system 200 may be constructed as a WEB server, ASP server,or the like, and the hardware structure thereof may include acommercially available information processing apparatus, such as aworkstation or a personal computer, and its accessories. Individualfunctions of the external system 200 are implemented by a CPU, a diskdrive, a memory unit, an input unit, an output unit, a communicationcontrol unit, etc., and programs for controlling them in the hardwarestructure of the external system 200.

In FIG. 13, the base sequence processing apparatus 100 schematicallyincludes a controller 102, such as a CPU, which controls the basesequence processing apparatus 100 overall; a communication controlinterface 104 which is connected to a communication device (not shown inthe drawing), such as a router, connected to a communication line or thelike; an input-output control interface 108 connected to an input unit112 and an output unit 114; and a memory 106 which stores variousdatabases and tables. These parts are connected via given communicationchannels in a communicable manner. Furthermore, the base sequenceprocessing apparatus 100 is connected to the network 300 in acommunicable manner via a communication device, such as a router, and awired or radio communication line.

Various databases and tables (a target gene base sequence file 106 a˜atarget gene annotation database 106 h) which are stored in the memory106 are storage means, such as fixed disk drives, for storing variousprograms used for various processes, tables, files, databases, files forweb pages, etc.

Among these components of the memory 106, the target gene base sequencefile 106 a is target gene base sequence storage means for storing basesequence information of the target gene for RNA interference. FIG. 14 isa diagram which shows an example of information stored in the targetgene base sequence file 106 a.

As shown in FIG. 14, the information stored in the target gene basesequence file 106 a consists of base sequence identification informationwhich uniquely identifies base sequence information of the target genefor RNA interference (e.g., “NM_(—)000507” in FIG. 14) and base sequenceinformation (e.g., “ATGGCTGA . . . AGTGA” in FIG. 14), the base sequenceidentification information and the base sequence information beingassociated with each other.

Furthermore, a partial base sequence file 106 b is partial base sequencestorage means for storing partial base sequence information, i.e., asequence segment having a predetermined number of bases in base sequenceinformation of the target gene for RNA interference. FIG. 15 is adiagram which shows an example of information stored in the partial basesequence file 106 b.

As shown in FIG. 15, the information stored in the partial base sequencefile 106 b consists of partial base sequence identification informationwhich uniquely identifies partial base sequence information (e.g.,“NM_(—)000507:36” in FIG. 15), partial base sequence information (e.g.,“caccct . . . tcatgg” in FIG. 15), and information on inclusion of anoverhanging portion which shows the inclusion of the overhanging portion(e.g., “included” in FIG. 15), the partial base sequence identificationinformation, the partial base sequence information, and the informationon inclusion of the overhanging portion being associated with eachother.

A determination result file 106 c is determination result storage meansfor storing the results determined by a 3′ end base determination part102 b, a 5′ end base determination part 102 c, and a predetermined baseinclusion determination part 102 d, which will be described below. FIG.16 is a diagram which shows an example of information stored in thedetermination result file 106 c.

As shown in FIG. 16, the information stored in the determination resultfile 106 c consists of partial base sequence identification information(e.g., “NM_(—)000507:36” in FIG. 16), determination result on 3′ endbase corresponding to a result determined by the 3′ end basedetermination part 102 b (e.g., “1” in FIG. 16), determination result on5′ end base corresponding to a result determined by the 5′ end basedetermination part 102 c (e.g., “1” in FIG. 16), determination result oninclusion of predetermined base corresponding to a result determined bythe predetermined base inclusion determination part 102 d (e.g., “4” inFIG. 16), and comprehensive determination result corresponding to aresult obtained by putting together the results determined by the 3′ endbase determination part 102 b, the 5′ end base determination part 102 c,and the predetermined base inclusion determination part 102 d (e.g., “4”in FIG. 16), the partial base sequence identification information, thedetermination result on 3′ end base, the determination result on 5′ endbase, the determination result on inclusion of predetermined base, andthe comprehensive determination result being associated with each other.

Additionally, FIG. 16 shows an example of the case in which, withrespect to the determination result on 3′ end base and the determinationresult on 5′ end base, “1” is set when determined as being “included” byeach of the 3′ end base determination part 102 b and the 5′ end basedetermination part 102 c and “0” is set when determined as being “notincluded”. Furthermore, FIG. 16 shows an example of the case in whichthe determination result on inclusion of predetermined base is set asthe number of bases corresponding to one or more types of bases selectedfrom the group consisting of adenine, thymine, and uracil contained inthe base sequence information comprising 7 bases at the 3′ end in thepartial base sequence information. Furthermore, FIG. 16 shows an exampleof the case in which the comprehensive determination result is set asthe product of the determination result on 3′ end base, thedetermination result on 5′ end base, and the determination result oninclusion of predetermined base. Specifically, for example, when theproduct is 3 or less, “0” may be set.

Furthermore, a prescribed sequence file 106 d is prescribed sequencestorage means for storing prescribed sequence information correspondingto partial base sequence information which specifically causes RNAinterference in the target gene. FIG. 17 is a diagram which shows anexample of information stored in the prescribed sequence file 106 d.

As shown in FIG. 17, the information stored in the prescribed sequencefile 106 d consists of partial base sequence identification information(e.g., “NM_(—)000507:36” in FIG. 17) and prescribed sequence informationcorresponding to partial base sequence information which specificallycauses RNA interference in the target gene (e.g., caccct . . . tcatgg”in FIG. 17), the partial base sequence identification information andthe prescribed sequence information being associated with each other.

Furthermore, a reference sequence database 106 e is a database whichstores reference base sequence information corresponding to basesequence information to which reference is made to search base sequenceinformation identical or similar to the prescribed sequence informationby an identical/similar base sequence search part 102 g, which will bedescribed below. The reference sequence database 106 e may be anexternal base sequence information database accessed via the Internet ormay be an in-house database created by copying such a database, storingthe original sequence information, or further adding unique annotationinformation to such a database. FIG. 18 is a diagram which shows anexample of information stored in the reference sequence database 106 e.

As shown in FIG. 18, the information stored in the reference sequencedatabase 106 e consists of reference sequence identification information(e.g., “ref|NM 015820.11” in FIG. 18) and reference base sequenceinformation (e.g., “caccct . . . gcatgg” in FIG. 18), the referencesequence identification information and the reference base sequenceinformation being associated with each other.

Furthermore, a degree of identity or similarity file 106 f is degree ofidentity or similarity storage means for storing the degree of identityor similarity corresponding to a degree of identity or similarity ofidentical or similar base sequence information searched by anidentical/similar base sequence search part 102 g, which will bedescribed below. FIG. 19 is a diagram which shows an example ofinformation stored in the degree of identity or similarity file 106 f.

As shown in FIG. 19, the information stored in the degree of identity orsimilarity file 106 f consists of partial base sequence identificationinformation (e.g., “NM_(—)000507:36” in FIG. 19), reference sequenceidentification information (e.g., “ref|NM 015820.11” and “ref|NM003837.11” in FIG. 19), and degree of identity or similarity (e.g.,“0.52” in FIG. 19), the partial base sequence identificationinformation, the reference sequence identification information, and thedegree of identity or similarity being associated with each other.

Furthermore, an evaluation result file 106 g is evaluation resultstorage means for storing the result of evaluation on whether genesunrelated to the target gene are targeted by an unrelated gene targetevaluation part 102 h, which will be described below. FIG. 20 is adiagram which shows an example of information stored in the evaluationresult file 106 g.

As shown in FIG. 20, the information stored in the evaluation resultfile 106 g consists of partial base sequence identification information(e.g., “NM_(—)000507:36” and “NM_(—)000507:441” in FIG. 20), total sumcalculated by a total sum calculation part 102 m, which will bedescribed below, (e.g., “5.9” and “170.8” in FIG. 20), and evaluationresult (e.g., “nontarget” and “target” in FIG. 20), the partial basesequence identification information, the total sum, and the evaluationresult being associated with each other. Additionally, in FIG. 20,“nontarget” means that the prescribed sequence information does nottarget genes unrelated to the target gene, and “target” means that theprescribed sequence information targets genes unrelated to the targetgene.

A target gene annotation database 106 h is target gene annotationstorage means for storing annotation information regarding the targetgene. The target gene annotation database 106 h may be an externalannotation database which stores annotation information regarding genesand which is accessed via the Internet or may be an in-house databasecreated by copying such a database, storing the original sequenceinformation, or further adding unique annotation information to such adatabase.

The information stored in the target gene annotation database 106 hconsists of target gene identification information which identifies thetarget gene (e.g., the name of a gene to be targeted, and Accessionnumber (e.g., “NM_(—)000507” and “FBP1” described on the top in FIG. 3))and simplified information on the target gene (e.g., “Homo sapiensfructose-1,6-bisphosphatase 1” describe on the top in FIG. 3), thetarget gene identification information and the simplified informationbeing associated with each other.

In FIG. 13, the communication control interface 104 controlscommunication between the base sequence processing apparatus 100 and thenetwork 300 (or a communication device, such as a router). Namely, thecommunication control interface 104 performs data communication withother terminals via communication lines.

In FIG. 13, the input-output control interface 108 controls the inputunit 112 and the output unit 114. Here, as the output unit 114, inaddition to a monitor (including a home television), a speaker may beused (hereinafter, the output unit 114 may also be described as amonitor). As the input unit 112, a keyboard, a mouse, a microphone, orthe like may be used. The monitor cooperates with a mouse to implement apointing device function.

In FIG. 13, the controller 102 includes control programs, such as OS(Operating System), programs regulating various processing procedures,etc., and internal memories for storing required data, and performsinformation processing for implementing various processes using theprograms, etc. The controller 102 functionally includes a partial basesequence creation part 102 a, a 3′ end base determination part 102 b, a5′ end base determination part 102 c, a predetermined base inclusiondetermination part 102 d, a prescribed sequence selection part 102 e, anoverhanging portion-adding part 102 f, an identical/similar basesequence search part 102 g, and an unrelated gene target evaluation part102 h.

Among them, the partial base sequence creation part 102a is partial basesequence creation means for acquiring base sequence information of atarget gene for RNA interference and creating partial base sequenceinformation corresponding to a sequence segment having a predeterminednumber of bases in the base sequence information. As shown in FIG. 21,the partial base sequence creation part 102 a includes a region-specificbase sequence creation part 102 i, a common base sequence creation part102 j, and an overhanging portion-containing base sequence creation part102 k.

FIG. 21 is a block diagram which shows an example of the structure ofthe partial base sequence creation part 102a of the system to which thepresent invention is applied and which shows only the parts related tothe present invention.

In FIG. 21, the region-specific base sequence creation part 102 i isregion-specific base sequence creation means for creating partial basesequence information having a predetermined number of bases from asegment corresponding to a coding region or transcription region of thetarget gene in the base sequence information.

The common base sequence creation part 102 j is common base sequencecreation means for creating partial base sequence information having apredetermined number of bases which is common in a plurality of basesequence information derived from different organisms.

The overhanging portion-containing base sequence creation part 102 k isoverhanging portion-containing base sequence creation means for creatingpartial base sequence information containing an overhanging portion.

Referring back to FIG. 13, the 3′ end base determination part 102 b is3′ end base determination means for determining whether the 3′ end basein the partial base sequence information is adenine, thymine, or uracil.

Furthermore, the 5′ end base determination part 102 c is 5′ end basedetermination means for determining whether the 5′ end base in thepartial base sequence information is guanine or cytosine.

Furthermore, the predetermined base inclusion determination part 102 dis predetermined base inclusion determination means for determiningwhether the base sequence information comprising 7 bases at the 3′ endin the partial base sequence information is rich in one or more types ofbases selected from the group consisting of adenine, thymine, anduracil.

Furthermore, the prescribed sequence selection part 102 e is prescribedsequence selection means for selecting prescribed sequence information,which specifically causes RNA interference in the target gene, from thepartial base sequence information based on the results determined by the3′ end base determination part 102 b, the 5′ end base determination part102 c, and the predetermined base inclusion determination part 102 c.

Furthermore, the overhanging portion-adding part 102 f is overhangingportion addition means for adding an overhanging portion to at least oneend of the prescribed sequence information.

Furthermore, the identical/similar base sequence search part 102 g isidentical/similar base sequence search means for searching base sequenceinformation, identical or similar to the prescribed sequenceinformation, from other base sequence information.

Furthermore, the unrelated gene target evaluation part 102 h isunrelated gene target evaluation means for evaluating whether theprescribed sequence information targets genes unrelated to the targetgene based on the identical or similar base sequence information. Asshown in FIG. 22, the unrelated gene target evaluation part 102 hfurther includes a total sum calculation part 102 m and a totalsum-based evaluation part 102 n.

FIG. 22 is a block diagram which shows an example of the structure ofthe unrelated gene target evaluation part 102 h of the system to whichthe present invention is applied and which schematically shows only theparts related to the present invention.

In FIG. 22, the total sum calculation part 102 m is total sumcalculation means for calculating the total sum of reciprocals of thevalues showing the degree of identity or similarity based on the totalamount of base sequence information on the genes unrelated to the targetgene in identical or similar base sequence information and the valuesshowing the degree of identity or similarity attached to the basesequence information on the genes unrelated to the target gene (identityor similarity).

Furthermore, the total sum-based evaluation part 102n is total sum-basedtarget evaluation means for evaluating whether the prescribed sequenceinformation targets genes unrelated to the target gene based on thetotal sum calculated by the total sum calculation part 102 m.

The details of processing of each part will be described later.

[Processing of the System]

An example of processing of the system having the configurationdescribed above in this embodiment will be described in detail withreference to FIGS. 23 and 24.

[Main Processing]

First, the details of the main processing will be described withreference to FIG. 23, etc. FIG. 23 is a flowchart which shows an exampleof the main processing of the system in this embodiment.

The base sequence processing apparatus 100 acquires base sequenceinformation of a target gene for RNA interference by the partial basesequence creation process performed by the partial base sequencecreation part 102 a, stores it in a predetermined memory region of thetarget gene base sequence file 106 a, creates partial base sequenceinformation corresponding to a sequence segment having a predeterminednumber of bases in the base sequence information, and stores the createdpartial base sequence information in a predetermined memory region ofthe partial base sequence file 106 b (step SA-1).

In step SA-1, the partial base sequence creation part 102 a may createpartial base sequence information having a predetermined number of basesfrom a segment corresponding to a coding region or transcription regionof the target gene in the base sequence information by the processing ofthe region-specific base sequence creation part 102 i and may store thecreated partial base sequence information in a predetermined memoryregion of the partial base sequence file 106 b.

In step SA-1, the partial base sequence creation part 102 a may createpartial base sequence information having a predetermined number of baseswhich is common in a plurality of base sequence information derived fromdifferent organisms (e.g., human base sequence information and mousebase sequence information) by the processing of the common base sequencecreation part 102 j and may store the created partial base sequenceinformation in a predetermined memory region of the partial basesequence file 106 b. Furthermore, common partial base sequenceinformation having a predetermined number of bases which is common in aplurality of analogous base sequence information in the same species maybe created.

In step SA-1, the partial base sequence creation part 102 a may createpartial base sequence information having a predetermined number of basesfrom segments corresponding to coding regions or transcription regionsof the target gene in a plurality of base sequence information derivedfrom different species by the processing of the region-specific basesequence creation part 102 i and the common base sequence creation part102 j and may store the created partial base sequence information in apredetermined memory region of the partial base sequence file 106 b.Furthermore, common partial base sequence information having apredetermined number of bases may be created from segments correspondingto coding regions or transcription regions of the target gene in aplurality of analogous base sequence information in the same species.

Furthermore, in step SA-1, the partial base sequence creation part 102 amay create partial base sequence information containing an overhangingportion by the processing of the overhanging portion-containing basesequence creation part 102 k. Specifically, for example, the partialbase sequence creation part 102 a may create partial base sequenceinformation to which the overhanging portion inclusion information whichshows the inclusion of the overhanging portion by the processing of theoverhanging portion-containing base sequence creation part 102 k and maystore the created partial base sequence information and the overhangingportion inclusion information so as to be associated with each other ina predetermined memory region of the partial base sequence file 106 b.

The upper limit of the predetermined number of bases is, in the case ofnot including the overhanging portion, preferably 28 or less, morepreferably 22 or less, and still more preferably 20 or less, and in thecase of including the overhanging portion, preferably 32 or less, morepreferably 26 or less, and still more preferably 24 or less. The lowerlimit of the predetermined number of bases is, in the case of notincluding the overhanging portion, preferably at least 13, morepreferably at least 16, and still more preferably at least 18, and inthe case of including the overhanging portion, preferably at least 17,more preferably at least 20, and still more preferably at least 22. Mostpreferably, the predetermined number of bases is, in the case of notincluding the overhanging portion, 19, and in the case of including theoverhanging portion, 23.

Subsequently, the base sequence processing apparatus 100 determineswhether the 3′ end base in the partial base sequence information createdin step SA-1 is adenine, thymine, or uracil by the processing of the 3′end base determination part 102 b and stores the determination result ina predetermined memory region of the determination result file 106 c(step SA-2). Specifically, for example, the base sequence processingapparatus 100 may store “1” when the 3′ end base in the partial basesequence information created in step SA-1 is adenine, thymine, oruracil, by the processing of the 3′ end base determination part 102 b,and “0” when it is not, in a predetermined memory region of thedetermination result file 106 c.

Subsequently, the base sequence processing apparatus 100 determineswhether the 5′ end base in the partial base sequence information createdin step SA-1 is guanine or cytosine by the processing of the 5′ end basedetermination part 102 c and stores the determination result in apredetermined memory region of the determination result file 106 c (stepSA-3). Specifically, for example, the base sequence processing apparatus100 may store “1” when the 5′ end base in the partial base sequenceinformation created in step SA-1 is guanine or cytosine, by theprocessing of the 5′ end base determination part 102 c, and “0” when itis not, in a predetermined memory region of the determination resultfile 106 c.

Subsequently, the base sequence processing apparatus 100 determineswhether the base sequence information comprising 7 bases at the 3′ endin the partial base sequence information created in step SA-1 is rich inone or more types of bases selected from the group consisting ofadenine, thymine, and uracil by the processing of the predetermined baseinclusion determination part 102 d and stores the determination resultin a predetermined memory region of the determination result file 106 c(step SA-4). Specifically, for example, the base sequence processingapparatus 100, by the processing of the predetermined base inclusiondetermination part 102 d, may store the number of bases corresponding toone or more types of bases selected from the group consisting ofadenine, thymine, and uracil contained in the base sequence informationcomprising 7 bases at the 3′ end in the partial base sequenceinformation created in step SA-1 in a predetermined memory region of thedetermination result file 106 c. The rule of determination in step SA-4regulates that base sequence information in the vicinity of the 3′ endof the partial base sequence information created in step SA-1 contains arich amount of one or more types of bases selected from the groupconsisting of adenine, thymine, and uracil, and more specifically, as anindex for search, regulates that the base sequence information in therange from the 3′ end base to the seventh base from the 3′ end is richin one or more types of bases selected from the group consisting ofadenine, thymine, and uracil.

In step SA-4, the phrase “base sequence information rich in” correspondsto the phrase “sequence rich in” described in the column <1> Method forsearching target base sequence for RNA interference. Specifically, forexample, when the partial base sequence information created in step SA-1comprises about 19 bases, in the base sequence information comprising 7bases at the 3′ end in the partial base sequence information, preferablyat least 3 bases, more preferably at least 4 bases, and particularlypreferably at least 5 bases, are one or more types of bases selectedfrom the group consisting of adenine, thymine, and uracil.

Furthermore, in steps SA-2 to SA-4, when partial base sequenceinformation including the overhanging portion is determined, thesequence segment excluding the overhanging portion in the partial basesequence information is considered as the determination target.

Subsequently, based on the determination results in steps SA-2, SA-3,and SA-4, the base sequence processing apparatus 100, by the processingof the prescribed sequence selection part 102 e, selects prescribedsequence information which specifically causes RNA interference in thetarget gene from the partial base sequence information created in stepSA-1 and stores it in a predetermined memory region of the prescribedsequence file 106 d (Step SA-5).

Specifically; for example, the base sequence processing apparatus 100,by the processing of the prescribed sequence selection part 102 e,selects partial base sequence information, in which the 3′ end base hasbeen determined as adenine, thymine, or uracil in step SA-2, the 5′ endbase has been determined as guanine or cytosine in step SA-3, and basesequence information comprising 7 bases at the 3′ end in the partialbase sequence information has been determined as being rich in one ormore types of bases selected from the group consisting of adenine,thymine, and uracil, as prescribed sequence information, and stores itin a predetermined memory region of the prescribed sequence file 106 d.Specifically, for example, the base sequence processing apparatus 100,by the processing of the prescribed sequence selection part 102 e, maycalculate a product of the values outputted in steps SA-2, SA-3, andSA-4 and, based on the product, select prescribed sequence informationfrom the partial base sequence information created in step SA-1.

Here, the base sequence processing apparatus 100 may add anoverhangingportion to at least one end of the prescribed sequenceinformation selected in step SA-5 by the processing of the overhangingportion-adding part 102 f, and may store it in a predetermined memoryregion of the prescribed sequence file 106 d. Specifically, for example,by the processing of the overhanging portion-adding part 102f, the basesequence processing apparatus 100 may change the prescribed sequenceinformation stored in the prescribed sequence information section in theprescribed sequence file 106 d to prescribed sequence information inwhich an overhanging portion is added to at least one end. Additionally,for example, when a target is searched, the overhanging portion may beadded to both ends of the prescribed sequence information.

Additionally, the number of bases in the overhanging portion correspondsto the number of bases described in the column <2> Method for designingbase sequence of polynucleotide for causing RNA interference.Specifically, for example, 2 is particularly suitable as the number ofbases.

Furthermore, the base sequence processing apparatus 100, by theprocessing of the identical/similar base sequence search part 102 g, maysearch base sequence information that is identical or similar to theprescribed sequence information selected in step SA-5 from other basesequence information (e.g., base sequence information published in apublic database, such as RefSeq of NCBI) using a known homology searchmethod, such as BLAST, FASTA, or ssearch, and based on the searchedidentical or similar base sequence information, by the unrelated genetarget evaluation process performed by the unrelated gene targetevaluation part 102 h, may evaluate whether the prescribed sequenceinformation targets genes unrelated to the target gene.

Specifically, for example, the base sequence processing apparatus 100,by the processing of the identical/similar base sequence search part 102g, may search base sequence information that is identical or similar tothe prescribed sequence information selected in step SA-5 from otherbase sequence information (e.g., base sequence information published ina public database, such as RefSeq of NCBI) using a known homology searchmethod, such as BLAST, FASTA, or ssearch. The unrelated gene targetevaluation part 102 h, by the processing of the total sum calculationpart 102 m, may calculate the total sum of the reciprocals of the valuesshowing the degree of identity or similarity based on the total amountof base sequence information on the genes unrelated to the target genein the searched identical or similar base sequence information and thevalues showing the degree of identity or similarity (e.g., “E value” inBLAST, FASTA, or ssearch) attached to the base sequence information onthe genes unrelated to the target gene. The unrelated gene targetevaluation part 102 h, by the processing of the total sum-basedevaluation part 102 n, may evaluate whether the prescribed sequenceinformation targets genes unrelated to the target gene based on thecalculated total sum.

Here, the details of the unrelated gene target evaluation processperformed by the unrelated gene target evaluation part 102 h will bedescribed with reference to FIG. 24.

FIG. 24 is a flowchart which shows an example of the unrelated geneevaluation process of the system in this embodiment.

First, the base sequence processing apparatus 100, by the processing ofthe identical/similar base sequence search part 102 g, searches basesequence information that is identical or similar to the prescribedsequence information selected in step SA-5 from other base sequenceinformation (e.g., base sequence information published in a publicdatabase, such as RefSeq of NCBI) using a known homology search method,such as BLAST, FASTA, or ssearch, and stores identification informationof the prescribed sequence information (“partial base sequenceidentification information” in FIG. 19), identification information ofthe searched identical or similar base sequence information (“referencesequence identification information” in FIG. 19), and the value showingthe degree of identity or similarity (e.g., “E value” in BLAST, FASTA,or ssearch) (“degree of identity or similarity” in FIG. 19) attached tothe searched identical or similar base sequence information so as to beassociated with each other in a predetermined memory region of thedegree of identity or similarity file 106f.

Subsequently, the unrelated gene target evaluation part 102 h, by theprocessing of the total sum calculation part 102 m, calculates the totalsum of reciprocals of the values showing the degree of identity orsimilarity based on the total amount of base sequence information on thegenes unrelated to the target gene in the searched identical or similarbase sequence information and the values showing the degree of identityor similarity (e.g., “E value” in BLAST, FASTA, or ssearch) attached tothe base sequence information on the genes unrelated to the target gene,and stores identification information of the prescribed sequenceinformation (“partial base sequence identification information” in FIG.20) and the calculated total sum (“total sum” in FIG. 20) so as to beassociated with each other in a predetermined memory region of theevaluation result file 106 g (step SB-1).

Subsequently, the unrelated gene target evaluation part 102 h, by theprocessing of the total sum-based evaluation part 102 n, evaluateswhether the prescribed sequence information targets genes unrelated tothe target gene based on the total sum calculated in step SB-1 (e.g.,based on the size of the total sum calculated in step SB-1), and storesthe evaluation results (“nontarget” and “target” in FIG. 20) in apredetermined memory region of the evaluation result file 106g (StepSB-2).

The main process is thereby completed.

Other Embodiments

One preferred embodiment of the present invention has been describedabove. However, it is to be understood that the present invention can becarried out in various embodiments other than the embodiment describedabove within the scope of the technical idea described in the claims.

For example, although the case in which the base sequence processingapparatus 100 performs processing on a stand-alone mode has beendescribed, construction may be made such that processing is performed inaccordance with the request from a client terminal which is constructedseparately from the base sequence processing apparatus 100, and theprocessing results are sent back to the client terminal. Specifically,for example, the client terminal transmits a name of the target gene forRNA interference (e.g., gene name or accession number) or base sequenceinformation regarding the target gene to the base sequence processingapparatus 100, and the base sequence processing apparatus 100 performsthe processes described above in the controller 102 on base sequenceinformation corresponding to the name or the base sequence informationtransmitted from the client terminal to select prescribed sequenceinformation which specifically causes RNA interference in the targetgene and transmits it to the client terminal. In such a case, forexample, by acquiring sequence information from a public database, siRNAagainst the gene in query may be selected. Alternatively, for example,siRNA for all the genes may be calculated and stored preliminarily, andsiRNA may be immediately selected in response to the request from theclient terminal (e.g., gene name or accession number) and the selectedsiRNA may be sent back to the client terminal.

Furthermore, the base sequence processing apparatus 100 may check thespecificity of prescribed sequence information with respect to genesunrelated to the target gene. Thereby, it is possible to selectprescribed sequence information which specifically causes RNAinterference only in the target gene.

Furthermore, in the system comprising a client terminal and the basesequence processing apparatus 100, an interface function may beintroduced in which, for example, the results of RNA interference effectof siRNA (e.g., “effective” or “not effective”) are fed back from theWeb page users on the Web, and the experimental results fed back fromthe users are accumulated in the base sequence processing apparatus 100so that the sequence regularity of siRNA effective for RNA interferenceis improved.

Furthermore, the base sequence processing apparatus 100 may calculatebase sequence information of a sense strand of siRNA and base sequenceinformation of an antisense strand complementary to the sense strandfrom the prescribed sequence information. Specifically, for example,when “caccctgacccgcttcgtcatgg” is selected as 23-base sequenceinformation wherein 2-base overhanging portions are added to both endsof the prescribed sequence as a result of the processes described above,the base sequence processing apparatus 100 calculates the base sequenceinformation of a sense strand “5′-CCCUGACCCGCUUCGUCAUGG-3′″ and the basesequence information of an antisense strand“5′-AUGACGAAGCGGGUCAGGGUG-3′″. Consequently, it is not necessary tomanually arrange the sense strand and the antisense strand when apolynucleotide is ordered, thus improving convenience.

Furthermore, in the processes described in the embodiment, the processesdescribed as being automatically performed may be entirely or partiallyperformed manually, or the processes described as being manuallyperformed may be entirely or partially performed automatically by aknown method.

In addition, processing procedures, control procedures, specific names,information including various registration data and parameters, such assearch conditions, examples of display screen, and database structuresmay be changed in any manner except when otherwise described.

Furthermore, with respect to the base sequence processing apparatus 100,the components are shown in the drawings only based on the functionalconcept, and it is not always necessary to physically construct thecomponents as shown in the drawings.

For example, the process functions of the individual parts or individualunits of the base sequence processing apparatus 100, in particular, theprocess functions performed in the controller 102, may be entirely orpartially carried out by a CPU (Central Processing Unit) or programswhich are interpreted and executed by the CPU. Alternatively, it may bepossible to realize the functions based on hardware according to a wiredlogic. Additionally, the program is recorded in a recording medium whichwill be described below and is mechanically read by the base sequenceprocessing apparatus 100 as required.

Namely, the memory 106, such as a ROM or HD, records a computer programwhich, together with OS (Operating System), gives orders to the CPU toperform various types of processing. The computer program is executed bybeing loaded into a RAM or the like, and, together with the CPU,constitutes the controller 102. Furthermore, the computer program may berecorded in an application program server which is connected to the basesequence processing apparatus 100 via any network 300, and may beentirely or partially downloaded as required.

The program of the present invention may be stored in acomputer-readable recording medium. Here, examples of the “recordingmedium” include any “portable physical medium”, such as a flexible disk,an optomagnetic disk, a ROM, an EPROM, an EEPROM, a CD-ROM, a MO, a DVD,or a flash disk; any “fixed physical medium”, such as a ROM, a RAM, or aHD which is incorporated into various types of computer system; and a“communication medium” which holds the program for a short period oftime, such as a communication line or carrier wave, in the case when theprogram is transmitted via a network, such as a LAN, a WAN, or Internet.

Furthermore, the “program” means a data processing method described inany language or by any description method, and the program may have anyformat (e.g., source code or binary code). The “program” is not alwayslimited to the one having a single system configuration, and may have adistributed system configuration including a plurality of modules orlibraries, or may achieve its function together with another program,such as OS (Operating System). With respect to specific configurationsand procedures for reading the recording medium in the individual unitsshown in the embodiment, or installation procedures after reading, etc.,known configurations and procedures may be employed.

The various types of databases, etc. (target gene base sequence file 106a˜target gene annotation database 106 h) stored in the memory 106 arestorage means, such as memories (e.g., RAMs and ROMs), fixed disk drives(e.g., hard disks), flexible disks, and optical disks, which storevarious types of programs used for various processes and Web siteprovision, tables, files, databases, files for Web pages, etc.

Furthermore, the base sequence processing apparatus 100 may be producedby connecting peripheral apparatuses, such as a printer, a monitor, andan image scanner, to a known information processing apparatus, forexample, an information processing terminal, such as a personal computeror a workstation, and installing software (including programs, data,etc.) which implements the method of the present invention into theinformation processing apparatus.

Furthermore, specific modes of distribution/integration of the basesequence processing apparatus 100, etc. are not limited to those shownin the specification and the drawings, and the base sequence processingapparatus 100, etc., may be entirely or partially distributed/integratedfunctionally or physically in any unit corresponding to various types ofloading, etc. (e.g., grid computing). For example, the individualdatabases may be independently constructed as independent databaseunits, or processing may be partially performed using CGI (CommonGateway Interface).

Furthermore, the network 300 has a function of interconnecting betweenthe base sequence processing apparatus 100 and the external system 200,and for example, may include any one of the Internet, intranets, LANs(including both wired and radio), VANs, personal computer communicationnetworks, public telephone networks (including both analog and digital),dedicated line networks (including both analog and digital), CATVnetworks, portable line exchange networks/portable packet exchangenetworks of the IMT2000 system, CSM system, or PDC/PDC-P system, radiopaging networks, local radio networks, such as the Bluetooth, PHSnetworks, and satellite communication networks, such as CS, BS, andISDB. Namely, the present system can transmit and receive various typesof data via any network regardless of wired or radio.

EXAMPLES

The present invention will be described in more detail with reference tothe examples. However, it is to be understood that the present inventionis not restricted by the examples.

Example 1

<1> Gene for Measuring RNAi Effect and Expression Vector

As a target gene for measuring an RNAi effect by siRNA, a firefly(Photinus pyralis, P. pyralis) luciferase (luc) gene (P. pyralis lucgene: accession number: U47296) was used, and as an expression vectorcontaining this gene, a pGL3-Control Vector (manufactured by PromegaCorporation) was used. The segment of the P. pyralis luc gene is locatedbetween an SV40 promoter and a poly A signal within the vector. As aninternal control gene, a luc gene of sea pansy (Renilla reniformis, R.reniformis) was used, and as an expression vector containing this gene,pRL-TK (manufactured by Promega Corporation) was used.

<2> Synthesis of 21-base Double-Stranded RNA (siRNA)

Synthesis of 21-base sense strand and 21-base antisense strand RNA(located as shown in FIG. 9; a to p) was entrusted to Genset Corporationthrough Hitachi Instrument Service Co., Ltd.

The double-stranded RNA used for inhibiting expression of the P. pyralisluc gene was prepared by associating sense and antisense strands. In theassociation process, the sense strand RNA and the antisense strand RNAwere heated for 3 minutes in a reaction liquid of 10 mM Tris-HCl (pH7.5) and 20 mM NaCl, incubated for one hour at 37° C., and left to standuntil the temperature reached room temperature. Formation ofdouble-stranded polynucleotides was assayed by electrophoresis on 2%agarose gel in a TBE buffer, and it was confirmed that almost all thesingle-stranded polynucleotides were associated to form double-strandedpolynucleotides.

<3> Mammalian Cell Cultivation

As mammalian cultured cells, human HeLa cells and HEK293 cells andChinese hamster CHO-KI cells (RIKEN Cell bank) were used. As a medium,Dulbecco's modified Eagle's medium (manufactured by Gibco BRL) to whicha 10% inactivated fetal bovine serum (manufactured by Mitsubishi Kasei)and as antibiotics, 10 units/ml of penicillin (manufactured by Meiji)and 50 μg/ml of streptomycin (manufactured by Meiji) had been added wasused. Cultivation was performed at 37° C. in the presence of 5% CO₂.

<4> Transfection of Target Gene, Internal Control Gene, and siRNA IntoMammalian Cultured Cells

The mammalian cells were seeded at a concentration of 0.2 to 0.3×10⁶cells/ml into a 24-well plate, and after one day, using a Ca-phosphateprecipitation method (Saibo-Kogaku Handbook (Handbook for cellengineering), edited by Toshio Kuroki et al., Yodosha (1992)), 1.0 μg ofpGL3-Control DNA, 0.5 or 1.0 μg of pRL-TK DNA, and 0.01, 0.1, 1, 10 or100 nM of siRNA were introduced.

<5> Drosophila Cell Cultivation

As drosophila cultured cells, S2 cells (Schneider, I., et al., J.Embryol. Exp. Morph., 27, 353-365 (1972)) were used. As a medium,Schneider's Drosophila medium (manufactured by Gibco BRL) to which a 10%inactivated fetal bovine serum (manufactured by Mitsubishi Kasei) and asantibiotics, 10 units/ml of penicillin (manufactured by Meiji) and 50μg/ml of streptomycin (manufactured by Meiji) had been added was used.Cultivation was performed at 25° C. in the presence of 5% CO₂.

<6> Transfection of Target Gene, Internal Control Gene, and siRNA IntoDrosophila Cultured Cells

The S2 cells were seeded at a concentration of 1.0×10⁶ cells/ml into a24-well plate, and after one day, using a Ca-phosphate precipitationmethod (Saibo-Kogaku Handbook (Handbook for cell engineering), edited byToshio Kuroki et al., Yodosha (1992)), 1.0 μg of pGL3-Control DNA, 0.1μg of pRL-TK DNA, and 0.01, 0.1, 1, 10 or 100 nM of siRNA wereintroduced.

<7> Measurement of RNAi Effect

The cells transfected with siRNA were recovered 20 hours aftertransfection, and using a Dual-Luciferase Reporter Assay System(manufactured by Promega Corporation), the levels of expression(luciferase activities) of two types of luciferase (P. pyralis luc andreniformis luc) protein were measured. The amount of luminescence wasmeasured using a Lumat LB9507 luminometer (EG&G Berthold).

<8> Results

The measurement results on the luciferase activities are shown in FIG.10. Furthermore, the results of study on correspondence between theluciferase activities and the individual base sequences are shown inFIG. 11.

In FIG. 10, the graph represented by B shows the results in thedrosophila cells, and the graph represented by C shows the results inthe human cells. As shown in FIG. 10, in the drosophila cells, bycreating RNA with a base number of 21, it was possible to inhibit theluciferase activities in almost all the sequences. On the other hand, inthe human cells, it was evident that it was difficult to obtainsequences which could inhibit the luciferase activities simply bysetting the base number at 21.

Analysis was then conducted on the regularity of base sequence withrespect to RNA a to p. As shown in FIG. 11, with respect to 5 points ofthe double-stranded RNA, the base sequence was analyzed. With respect tosiRNA a in the top row of the table shown in FIG. 11, the relativeluciferase activity (RLA) is 0.03. In the antisense strand, from the 3′end, the base sequence of the overhanging portion (OH) is UC; the G/Ccontent (content of guanine or cytosine) in the subsequent 7 bases (3′-Tin FIG. 11) is 57%; the G/C content in the further subsequent 5 bases (Min FIG. 11) is 20; the G/C content in the further subsequent 7 bases(5′-T in FIG. 11) is 14%; the 5′ end is U; and the G/C content in totalis 32%. In the table, a lower RLA value indicates lower RLA activity,i.e., inhibition of the expression of luciferase.

As is evident from the results, in the base sequence of polynucleotidesfor causing RNA interference, it is highly probable that the 3′ end isadenine or uracil and that the 5′ end is guanine or cytosine.Furthermore, it has become clear that the 7-base sequence from the 3′end is rich in adenine or uracil.

Example 21

1. Construction of Target Expression Vector pTREC

A target expression vector was constructed as follows. A targetexpression molecule is a molecule which allows expression of RNA havinga sequence to be targeted by RNAi (hereinafter, also referred to as a“target sequence”).

A target mRNA sequence was constructed downstream of the CMVenhancer/promoter of pCI-neo (GenBank Accession No. U47120, manufacturedby Promega.Corporation) (FIG. 25). That is, the followingdouble-stranded oligomer was synthesized, the oligomer including a Kozaksequence (Kozak), an ATG sequence, a cloning site having a 23 base-pairsequence to be targeted (target), and an identification sequence forrestriction enzyme (NheI, EcoRI, XhoI) for recombination. Thedouble-stranded oligomer consists of a sequence shown in SEQ ID NO: 1 inthe sequence listing and its complementary sequence. The synthesizeddouble-stranded oligomer was inserted into the NheI/XbaI site of thepCI-neo to construct a target expression vector pTREC (FIG. 25). Withrespect to the intron, the intron site derived from β-globin originallyincorporated in the pCI-neo was used. (SEQ ID NO:1)5′-gctagccaccatggaattcacgcgtctcgagtctaga-3′

The pTREC shown in FIG. 25 is provided with a promoter and an enhancer(pro/enh) and regions PAR(F) 1 and PAR(R) 1 corresponding to the PCRprimers. An intron (Intron) is inserted into PAR(F) 1, and theexpression vector is designed such that the expression vector itselfdoes not become a template of PCR. After transcription of RNA, in anenvironment in which splicing is performed in eukaryotic cultured cellsor the like, the intron site of the pTREC is removed to join twoneighboring PAR(F) 1's. RNA produced from the pTREC can be amplified byRT-PCR. With respect to the intron, the intron site derived fromβ-globin originally incorporated in the pCI-neo was used.

The pTREC is incorporated with a neomycin-resistant gene (neo) as acontrol, and by preparing PCR primers corresponding to a part of thesequence in the neomycin-resistant gene and by subjecting the part ofthe neomycin-resistant gene to RT-PCR, the neomycin-resistant gene canbe used as an internal standard control (internal control). PAR(F) 2 andPAR(R) 2 represent the regions corresponding to the PCR primers in theneomycin-resistant gene. Although not shown in the example of FIG. 25,an intron may be inserted into at least one of PAR(F) 2 and PAR(R) 2.

2. Effect of Primer for Detecting Target mRNA

(1) Transfection Into Cultured Cells

HeLa cells were seeded at 0.2 to 0.3×10⁶ cells per well of a 24-wellplate, and after one day, using Lipofectamine 2000 (manufactured byInvitrogen Corp.), 0.5 μg of pTREC vector was transfected according tothe manual.

(2) Recovery of Cells and Quantification of mRNA

One day after the transfection, the cells were recovered and total RNAwas extracted with Trizol (manufactured by Invitrogen Corp.). Onehundred nanograms of the resulting RNA was reverse transcribed bySuperScript II RT (manufactured by Invitrogen Corp.), using oligo (dT)primers, to synthesize cDNA. A control to which no reverse transcriptasewas added was prepared. Using one three hundred and twentieth of theamount of the resulting cDNA as a PCR template, quantitative PCR wascarried out in a 50-μl reaction system using SYBR Green PCR Master Mix(manufactured by Applied Biosystems Corp.) to quantify target mRNA(referred to as mRNA (T)) and, as an internal control, mRNA derived fromthe neomycin-resistant gene in the pTREC (referred to as mRNA (C)). Areal-time monitoring apparatus ABI PRIZM7000 (manufactured by AppliedBiosystems) was used for the quantitative PCR. A primer pair T (SEQ IDNOs: 2 and 3 in the sequence listing) and a primer pair C (SEQ ID NOs: 4and 5 in the sequence listing) were used for the quantification of mRNA(T) and mRNA (C), respectively. Primer pair T: aggcactgggcaggtgtc (SEQID NO:2) tgctcgaagcattaaccctcacta (SEQ ID NO:3) Primer pair Catcaggatgatctggacgaag (SEQ ID NO:4) ctcttcagcaatatcacgggt (SEQ ID NO:5)

FIGS. 26 and 27 show the results of PCR. Each of FIGS. 26 and 27 is agraph in which the PCR product is taken on the axis of ordinate and thenumber of cycles of PCR is taken on the axis of abscissa. In theneomycin-resistant gene, there is a small difference in theamplification of the PCR product between the case in which cDNA wassynthesized by the reverse transcriptase (+RT) and the control casewhich no reverse transcriptase was added (−RT) (FIG. 26). This indicatesthat not only cDNA but also the vector remaining in the cells also actedas a template and was amplified. On the other hand, in target sequencemRNA, there is a large difference between the case in which the reversetranscriptase was added (+RT) and the case in which no transcriptase wasadded (−RT) (FIG. 27). This result indicates that since one member ofthe primer pair T is designed so as to sandwich the intron, cDNA derivedfrom intron-free mRNA is efficiently amplified, while the remainingvector having the intron does not easily become a template.

3. Inhibition of Expression of Target mRNA by siRNA

(1) Cloning of Evaluation Sequence to Target Expression Vector

Sequences corresponding to the coding regions 812-834 and 35-57 of ahuman vimentin (VIM) gene (RefSeq ID: NM_(—)003380) were targeted forevaluation. The following synthetic oligonucleotides (evaluationsequence fragments) of SEQ ID NOs: 6 and 7 in the sequence listing wereproduced, the synthetic oligonucleotides including these sequences andidentification sequences for EcoRI and XhoI. Evaluation sequence VIM35(corresponding to 35-57 of VIM) (SEQ ID NO:6)5′-gaattcgcaggatgttcggcggcccgggcctcgag-3′

Evaluation sequence VIM812 (corresponding to 812-834 of VIM) (SEQ IDNO:7) 5′-gaattcacgtacgtcagcaatatgaaagtctcgag-3′

Using the EcoRI and XhoI sites located on both ends of each of theevaluation sequence fragments, each fragment was cloned as a new targetsequence between the EcoRI and XhoI sites of the pTREC, and therebypTREC-VIM35 and pTREC-VIM812 were constructed.

(2) Production of siRNA

siRNA fragments corresponding to the evaluation sequence VIM35 (SEQ IDNO: 8 in the sequence list, FIG. 28), the evaluation sequence VIM812(SEQ ID NO: 9, FIG. 29), and a control sequence (siContorol, SEQ ID NO:10, FIG. 30) were synthesized, followed by annealing. Each of thefollowing siRNA sequences is provided with an overhanging portion on the3′ end. siVIM35 5′-aggauguucggcggcccgggc-3′ (SEQ ID NO:8) siVIM8125′-guacgucagcaauaugaaagu-3′ (SEQ ID NO:9) As a control, siRNA for theluciferase gene was used. siControl 5′-cauucuauccgcuggaagaug-3′ (SEQ IDNO:10)(3) Transfection Into Cultured Cells

HeLa cells were seeded at 0.2 to 0.3×10⁶ cells per well of a 24-wellplate, and after one day, using Lipofectamine 2000 (manufactured byInvitrogen Corp.), 0.5 μg of pTREC-VIM35 or pTREC-VIM812, and 100 nM ofsiRNA corresponding to the sequence derived from each VIM (siVIM35,siVIM812) were simultaneously transfected according to the manual. Intothe control cells, 0.5 μg of pTREC-VIM35 or pTREC-VIM812 and 100 nM ofsiRNA for the luciferase gene (siControl) were simultaneouslytransfected.

(4) Recovery of Cells and Quantification of mRNA

One day after the transfection, the cells were recovered and total RNAwas extracted with Trizol (Invitrogen). One hundred nanograms of theresulting RNA was reverse transcribed by SuperScript II RT (manufacturedby Invitrogen Corp.), using oligo (dT) primers, to synthesize cDNA.Using one three hundred and twentieth of the amount of the resultingcDNA as a PCR template, quantitative PCR was carried out in a 50-μlreaction system using SYBR Green PCR Master Mix (manufactured by AppliedBiosystems Corp.) to quantify mRNA (referred to as mRNA (T)) includingthe sequence derived from VIM to be evaluated and, as an internalcontrol, mRNA derived from the neomycin-resistant gene in the pTREC(referred to as mRNA (C)).

A real-time monitoring apparatus ABI PRIZM7000 (manufactured by AppliedBiosystems) was used for the quantitative PCR. The primer pair T (SEQ IDNOs: 2 and 3 in the sequence listing) and the primer pair C (SEQ ID NOs:4 and 5 in the sequence listing) were used for the quantification ofmRNA (T) and mRNA (C), respectively. The ratio (T/C) of the resultingvalues of mRNA was taken on the axis of ordinate (relative amount oftarget mRNA (%)) in a graph (FIG. 31).

In the control cells, since siRNA for the luciferase gene does notaffect target mRNA, the ratio T/C is substantially 1. In VIM812 siRNA,the ratio T/C is extremely decreased. The reason for this is that VIM812siRNA cut mRNA having the corresponding sequence, and it was shown thatVIM812 siRNA has the RNAi effect. On the other hand, in VIM35 siRNA, theT/C ratio was substantially the same as that of the control, and thus itwas shown that the sequence of VIM35 does not substantially have theRNAi effect.

Example 3

1. Inhibition of Expression of Endogenous Vimentin by siRNA

(1) Transfection Into Cultured Cells

HeLa cells were seeded at 0.2 to 0.3×10⁶ cells per well of a 24-wellplate, and after one day, using Lipofectamine 2000 (manufactured byInvitrogen Corp.), 100 nM of siRNA for VIM (siVIM35 or siVIM812) orcontrol siRNA (siControl) and, as a control for transfection efficiency,0.5 μg of pEGFP (manufactured by Clontech) were simultaneouslytransfected according to the manual. pEGFP is incorporated with EGFP.

(2) Assay of Endogenous Vimentin mRNA

Three days after the transfection, the cells were recovered and totalRNA was extracted with Trizol (manufactured by Invitrogen Corp.). Onehundred nanograms of the resulting RNA was reverse transcribed bySuperScript II RT (manufactured by Invitrogen Corp.), using oligo (dT)primers, to synthesize cDNA. PCR was carried out using the cDNA productas a template and using primers for vimentin, VIM-F3-84 and VIM-R3-274(SEQ ID NOs: 11 and 12). VIM-F3-84; gagctacgtgactacgtcca (SEQ ID NO: 11)VIM-R3-274; gttcttgaactcggtgttgat (SEQ ID NO: 12) Furthermore, as acontrol, PCR was carried out using β-actin primers ACTB-F2-481 andACTB-R2-664 (SEQ ID NOs: 13 and 14). The level of expression of vimentinwas evaluated under the common quantitative value of β-actin for eachsample. ACTB-F2-481; cacactgtgcccatctacga (SEQ ID NO:13) ACTB-R2-664;gccatctcttgctcgaagtc (SEQ ID NO:14)

The results are shown in FIG. 32. In FIG. 32, the case in whichsiControl (i.e., the sequence unrelated to the target) is incorporatedis considered as 100% for comparison, and the degree of decrease in mRNAof VIM when siRNA is incorporated into VIM is shown. siVIM-812 was ableto effectively inhibit VIM mRNA. In contrast, use of siVIM-35 did notsubstantially exhibit the RNAi effect.

(3) Antibody Staining of Cells

Three days after the transfection, the cells were fixed with 3.7%formaldehyde, and blocking was performed in accordance with aconventional method. Subsequently, a rabbit anti-vimentin antibody(α-VIM) or, as an internal control, a rabbit anti-Yes antibody (α-Yes)was added thereto, and reaction was carried out at room temperature.Subsequently, the surfaces of the cells were washed with PBS (PhosphateBuffered Saline), and as a secondary antibody, a fluorescently-labeledanti-rabbit IgG antibody was added thereto. Reaction was carried out atroom temperature. After the surfaces of the cells were washed with PBS,observation was performed using a fluorescence microscope.

The fluorescence microscope observation results are shown in FIG. 33. Inthe nine frames of FIG. 33, the parts appearing white correspond tofluorescent portions. In EGFP and Yes, substantially the same expressionwas confirmed in all the cells. In the cells into which siControl andsiVIM35 were introduced, fluorescence due to antibody staining ofvimentin was observed, and the presence of endogenous vimentin wasconfirmed. On the other hand, in the cells into which siVIM812 wasintroduced, fluorescence was significantly weaker than that of the cellsinto which siControl and siVIM35 were introduced. The results show thatendogenous vimentin mRNA was interfered by siVIM812, and consequently,the level of expression of vimentin protein was decreased. It has becomeevident that siVIM812 also has the RNAi effect against endogenousvimentin mRNA.

The results obtained in the assay system of the present invention[Example 2] matched well with the results obtained in the cases in whichendogenous genes were actually treated with corresponding siRNA [Example3]. Consequently, it has been confirmed that the assay system iseffective as a method for evaluating the RNAi activity of any siRNA.

Example 4

Base sequences were designed based on the predetermined rules (a) to(d). The base sequences were designed by a base sequence processingapparatus which runs the siRNA sequence design program. As the basesequences, 15 sequences (SEQ ID NOs: 15 to 29) which were expected tohave RNAi activity and 5 sequences (SEQ ID NOs: 30 to 34) which were notexpected to have RNAi activity were prepared.

RNAi activity was evaluated by measuring the luciferase activity as inExample 1 except that the target sequence and siRNA to be evaluated wereprepared based on each of the designed sequences. The results are shownin FIG. 34. A low luciferase relative activity value indicates aneffective state, i.e., siRNA provided with RNAi activity. All of thesiRNA which was expected to have RNAi activity by the programeffectively inhibited the expression of luciferase.

[Sequences which exhibited RNAi activity; prescribed sequence portions,excluding overhanging portions]    5, gacgccaaaaacataaaga (SEQ ID NO:15) 184, gttggcagaagctatgaaa (SEQ ID NO:16)  272, gtgttgggcgcgttattta (SEQID NO:17)  309, ccgcgaacgacatttataa (SEQ ID NO:18)  428,ccaatcatccaaaaaatta (SEQ ID NO:19)  515, cctcccggttttaatgaat (SEQ IDNO:20)  658, gcatgccagagatcctatt (SEQ ID NO:21)  695,ccggatactgcgattttaa (SEQ ID NO:22)  734, ggttttggaatgtttacta (SEQ IDNO:23)  774, gatttcgagtcgtcttaat (SEQ ID NO:24)  891,gcactctgattgacaaata (SEQ ID NO:25)  904, caaatacgatttatctaat (SEQ IDNO:26) 1186, gattatgtccggttatgta (SEQ ID NO:27) 1306,ccgcctgaagtctctgatt (SEQ ID NO:28) 1586, ctcgacgcaagaaaaatca (SEQ IDNO:29)

[Sequences which did not exhibit RNAi activity; prescribed sequenceportions, excluding overhanging portions]   14, aacataaagaaaggcccgg (SEQID NO:30)  265, tatgccggtgttgggcgcg (SEQ ID NO:31)  295,agttgcagttgcgcccgcg (SEQ ID NO:32)  411, acgtgcaaaaaaagctccc (SEQ IDNO:33) 1044, ttctgattacacccgaggg (SEQ ID NO:34)

Example 5

siRNA sequences for the SARS virus were designed and the RNAi activitiesthereof were investigated. The RNAi activity was evaluated by the sameassay as used in Example 2 except that the target sequences and thesequences to be evaluated were changed.

The siRNA sequences were designed with respect to 3CL-PRO, RdRp, Spikeglycoprotein, Small envelope E protein, Membrane glycoprotein M,Nucleocapsid protein, and s2m motif from the genome of the SARS virus,using the siRNA sequence design program, so as to conform to thepredetermined regularity.

As a result of the assay shown in FIG. 35, 11 siRNA sequences designedso as to conform to the regularity effectively inhibited RNA in whichcorresponding siRNA sequences were incorporated as targets. The case inwhich siControl (the sequence unrelated to SARS) is incorporated isconsidered as being 100%, and the relative amount of target mRNA in thecase in which each siRNA sequence of SARS is incorporated is shown. Wheneach siRNA sequence is incorporated, the amount of target RNA wasdecreased to about 10% or less, and the presence of the RNAi activitywas confirmed.

[siRNA sequences designed (prescribed sequence portions, excludingoverhanging portions)]

siControl;gggcgcggtcggtaaagtt (SEQ ID NO: 35)

3CL-PRO;SARS-10754;ggaattgccgtcttagata (SEQ ID NO: 36)

3CL-PRO;SARS-10810;gaatggtcgtactatcctt (SEQ ID NO: 37)

RdRp;SARS-14841;ccaagtaatcgttaacaat (SEQ ID NO: 38)

Spike glycoprotein;SARS-23341;gcttggcgcatatattcta (SEQ ID NO: 39)

Spike glycoprotein;SARS-24375;cctttcgcgacttgataaa (SEQ ID NO: 40)

Small envelope E protein;SARS-26233;gtgcgtactgctgcaatat (SEQ ID NO: 41)

Small envelope E protein;SARS-26288;ctactcgcgtgttaaaaat (SEQ ID NO: 42)

Membrane glycoprotein M;SARS-26399;gcagacaacggtactatta (SEQ ID NO: 43)

Membrane glycoprotein M;SARS-27024;dcggtagcaacgacaatat (SEQ ID NO: 44)

Nucleocapsid protein;SARS-28685;cgtagtcgcggtaattcaa (SEQ ID NO: 45)

2m motif;SARS-29606;gatcgagggtacagtgaat (SEQ ID NO: 46)

Example 6

The following siRNA sequences were designed in accordance with thecolumns “<5> siRNA sequence design program” and “<7> Base sequenceprocessing apparatus for running siRNA sequence design program, etc.”.The designed siRNA sequences are shown under SEQ ID NOs: 47 to 892 inthe sequence listing.

(Target Gene of RNAi)

NM_(—)000604, Homo sapiens fibroblast growth factor receptor 1(fins-related tyrosine kinase 2, Pfeiffer syndrome) (FGFR1).

(Target Sequences)

NM_(—)000604-807,gtagcaacgtggagttcat (SEQ ID NO: 47)

NM_(—)000604-806,ggtagcaacgtggagttca (SEQ ID NO: 48)

NM_(—)000604-811,caacgtggagttcatgtgt (SEQ ID NO: 49)

NM_(—)000604-880,ggtgaatgggagcaagatt (SEQ ID NO: 50)

NM_(—)000604-891,gcaagattggcccagacaa (SEQ ID NO: 51)

(Target Sequence Effective for Mouse Homolog)

NM_(—)000604-818,gagttcatgtgtaaggtgt (SEQ ID NO: 52)

(Target Gene of RNAi)

NM_(—)000141, Homo sapiens fibroblast growth factor receptor 2(bacteria-expressed kinase, keratinocyte growth factor receptor,craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome,Jackson-Weiss syndrome) (FGFR2).

(Target Sequences)

NM′-000141-612,gaggctacaaggtacgaaa (SEQ ID NO: 53)

NM_(—)000141-615,gctacaaggtacgaaacca (SEQ ID NO: 54)

NM_(—)000141-637,ctggagcctcattatggaa (SEQ ID NO: 55)

NM_(—)000141-574,gaaaaacgggaaggagttt (SEQ ID NO: 56)

(Target Sequences Effective for Mouse Homolog)

NM_(—)000141-595,gcaggagcatcgcattgga (SEQ ID NO: 57)

NM_(—)000141-69,ccttcagtttagttgagga (SEQ ID NO: 58)

NM_(—)000141-70,cttcagtttagttgaggat (SEQ ID NO: 59)

(Target Gene of RNAi)

NM_(—)000142, Homo sapiens fibroblast growth factor receptor 3(achondroplasia, thanatophoric dwarfism) (FGFR3).

(Target Sequences)

NM_(—)000142-899,gacggcacaccctacgtta (SEQ ID NO: 60)

NM_(—)000142-1925,cacaacctcgactactaca (SEQ ID NO: 61)

NM_(—)000142-2154,gcacacacgacctgtacat (SEQ ID NO: 62)

NM_(—)000142-678,cctgcgtcgtggagaacaa (SEQ ID NO: 63)

NM_(—)000142-2157,cacacgacctgtacatgat (SEQ ID NO: 64)

(Target Sequence Effective for Mouse Homolog)

NM_(—)000142-812,gagttccactgcaaggtgt (SEQ ID NO: 65)

(Target Gene of RNAi)

NM_(—)004448, Homo sapiens v-erb-b2 erythroblastic leukemia viraloncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian)(ERBB2).

(Target Sequences)

NM_(—)004448-356,ggagacccgctgaacaata (SEQ ID NO: 66)

NM_(—)004448-3645,ccttcgacaacctctatta (SEQ ID NO: 67)

NM_(—)004448-3237,gggctggctccgatgtatt (SEQ ID NO: 68)

NM_(—)004448-3238,ggctggctccgatgtattt (SEQ ID NO: 69)

NM_(—)004448-3240,ctggctccgatgtatttga (SEQ ID NO: 70)

(Target Gene of RNAi)

NM_(—)001982, Homo sapiens v-erb-b2 erythroblastic leukemia viraloncogene homolog 3 (avian) (ERBB3).

(Target Sequences)

NM_(—)001982-1347,gtgctgggcgtatctatat (SEQ ID NO: 71)

NM_(—)001982-1349,gctgggcgtatctatataa (SEQ ID NO: 72)

NM_(—)001982-1548,gcttgtcctgtcgaaatta (SEQ ID NO: 73)

NM_(—)001982-1549,cttgtcctgtcgaaattat (SEQ ID NO: 74)

NM_(—)001982-2857,cattcgcccaacctttaaa (SEQ ID NO: 75)

(Target Gene of RNAi)

NM_(—)005235, Homo sapiens v-erb-a erythroblastic leukemia viraloncogene homolog 4 (avian) (ERBB4).

(Target Sequences)

NM_(—)005235-295,ggagaatttacgcattatt (SEQ ID NO: 76)

NM_(—)005235-2120,gctcaacttcgtattttga (SEQ ID NO: 77)

NM_(—)005235-2940,ctcaaagatacctagttat (SEQ ID NO: 78)

NM_(—)005235-2121,ctcaacttcgtattttgaa (SEQ ID NO: 79)

NM_(—)005235-2880,ctgacagtagacctaaatt (SEQ ID NO: 80)

(Target Gene of RNAi)

NM_(—)002227, Homo sapiens Janus kinase 1 (a protein tyrosine kinase)(JAK1).

(Target Sequences)

NM_(—)002227-441,ctcagggacagtatgattt (SEQ ID NO: 81)

NM_(—)002227-1299,cagaatacgccatcaataa (SEQ ID NO: 82)

NM_(—)002227-673,gatgcggataaataatgtt (SEQ ID NO: 83)

NM_(—)002227-672,ggatgcggataaataatgt (SEQ ID NO: 84)

NM_(—)002227-3385,ctttcagaaccttattgaa (SEQ ID NO: 85)

(Target Sequences Effective for Mouse Homolog)

NM_(—)002227-607,cagctacaagcgatatatt (SEQ ID NO: 86)

NM_(—)002227-3042,caattgaaaccgataagga (SEQ ID NO: 87)

NM_(—)002227-2944,gggttctcggcaatacgtt (SEQ ID NO: 88)

(Target Gene of RNAi)

NM_(—)004972, Homo sapiens Janus kinase 2 (a protein tyrosine kinase)(JAK2).

(Target Sequences)

NM_(—)004972-2757,ctggtcggcgtaatctaaa (SEQ ID NO: 89)

NM_(—)004972-2759,ggtcggcgtaatctaaaat (SEQ ID NO: 90)

NM_(—)004972-2760,gtcggcgtaatctaaaatt (SEQ ID NO: 91)

NM_(—)004972-3175,ggaatttatgcgtatgatt (SEQ ID NO: 92)

NM_(—)004972-1452,ctgttcgctcagacaatat (SEQ ID NO: 93)

(Target Sequences Effective for Mouse Homolog)

NM_(—)004972-872,ggaaacggtggaattcagt (SEQ ID NO: 94)

NM_(—)004972-870,ctggaaacggtggaattca (SEQ ID NO: 95)

NM_(—)004972-847,gatttttgcaaccattata (SEQ ID NO: 96)

(Target Gene of RNAi)

NM_(—)000215, Homo sapiens Janus kinase 3 (a protein tyrosine kinase,leukocyte) (JAK3).

(Target Sequences)

NM_(—)000215-2315,gtcattcgtgacctcaata (SEQ ID NO: 97)

NM_(—)000215-2522,gacccgctagcccacaata (SEQ ID NO: 98)

NM_(—)000215-2524,cccgctagcccacaataca (SEQ ID NO: 99)

NM_(—)000215-1788,ccatggtgcaggaatttgt (SEQ ID NO: 100)

NM_(—)000215-1825,catgtatctgcgaaaacgt (SEQ ID NO: 101)

(Target Gene of RNAi)

NM_(—)003331, Homo sapiens tyrosine kinase 2 (TYK2).

(Target Sequences)

NM_(—)003331-3213,gcctgaaggagtataagtt (SEQ ID NO: 102)

NM_(—)003331-2658,cggaccctacggttttcca (SEQ ID NO: 103)

NM_(—)003331-299,ctatatttccgcataaggt (SEQ ID NO: 104)

(Target Sequences Effective for Mouse Homolog)

NM_(—)003331-2674,ccacaagcgctatttgaaa (SEQ ID NO: 105)

NM_(—)003331-2675,cacaagcgctatttgaaaa (SEQ ID NO: 106)

NM_(—)003331-328,gaactggcatggcatgaat (SEQ ID NO: 107)

(Target Gene of RNAi)

NM_(—)001079, Homo sapiens zeta-chain (TCR) associated protein kinase 70kDa (ZAP70).

(Target Sequences)

NM_(—)001079-512,gaggccgagcgcaaacttt (SEQ ID NO: 108)

NM_(—)001079-1512,ggtacgcacccgaatgcat (SEQ ID NO: 109)

NM_(—)001079-242,gagctctgcgagttctact (SEQ ID NO: 110)

NM_(—)001079-929,gacacgagcgtgtatgaga (SEQ ID NO: 111)

NM_(—)001079-1412,cggcactacgccaagatca (SEQ ID NO: 112)

(Target Sequence Effective for Mouse Homolog)

NM_(—)001079-1566,ggagctatggggtcaccat (SEQ ID NO: 113)

(Target Gene of RNAi)

NM_(—)005417, Homo sapiens v-src sarcoma (Schmidt-Ruppin A-2) viraloncogene homolog (avian) (SRC).

(Target Sequences)

NM_(—)005417-185,ctgttcggaggcttcaact (SEQ ID NO: 114)

NM_(—)005417-685,ggtggcctactactccaaa (SEQ ID NO: 115)

NM_(—)005417-474,gggagtcagagcggttact (SEQ ID NO: 116)

NM_(—)005417-480,cagagcggttactgctcaa (SEQ ID NO: 117)

NM_(—)005417-567,cagtgtctgacttcgacaa (SEQ ID NO: 118)

(Target Sequence Effective for Mouse Homolog)

NM_(—)005417-651,cctcccgcacccagttcaa (SEQ ID NO: 119)

(Target Gene of RNAi)

NM_(—)002350, Homo sapiens v-yes-1 Yamaguchi sarcoma viral relatedoncogene homolog (LYN).

(Target Sequences)

NM_(—)002350-610,cagcgacatgattaaacat (SEQ ID NO: 120)

NM_(—)002350-533,gttattaagcactacaaaa (SEQ ID NO: 121)

NM_(—)002350-606,gtatcagcgacatgattaa (SEQ ID NO: 122)

(Target Sequences Effective for Mouse Homolog)

NM_(—)002350-783,ggatgggttactataacaa (SEQ ID NO: 123)

NM_(—)002350-694,gaagccatgggataaagat (SEQ ID NO: 124)

NM_(—)002350-541,gcactacaaaattagaagt (SEQ ID NO: 125)

(Target Gene of RNAi)

NM_(—)005157, Homo sapiens v-abl Abelson murine leukemia viral oncogenehomolog 1 (ABL1).

(Target Sequences)

NM_(—)005157-232,cactctaagcataactaaa (SEQ ID NO: 126)

NM_(—)005157-770,gagggcgtgtggaagaaat (SEQ ID NO: 127)

NM_(—)005157-262,ccgggtcttaggctataat (SEQ ID NO: 128)

NM_(—)005157-264,gggtcttaggctataatca (SEQ ID NO: 129)

NM_(—)005157-484,catctcgctgagatacgaa (SEQ ID NO: 130)

(Target Sequences Effective for Mouse Homolog)

NM_(—)005157-217,ggccagtggagataacact (SEQ ID NO: 131)

NM_(—)005157-1227,gcctggcctacaacaagtt (SEQ ID NO: 132)

NM_(—)005157-680,gtgtcccccaactacgaca (SEQ ID NO: 133)

(Target Gene of RNAi)

NM_(—)005158, Homo sapiens v-abl Abelson murine leukemia viral oncogenehomolog 2 (arg, Abelson-related gene) (ABL2).

(Target Sequences)

NM_(—)005158-3273,ctcaaactcgcaacaaatt (SEQ ID NO: 134)

NM_(—)005158-3272,cctcaaactcgcaacaaat (SEQ ID NO: 135)

NM_(—)005158-1425,ctaaggtttatgaacttat (SEQ ID NO: 136)

NM_(—)005158-448,gctcagcagtctaatcaat (SEQ ID NO: 137)

NM_(—)005158-3110,caggccgctgagaaaatct (SEQ ID NO: 138)

(Target Gene of RNAi)

NM_(—)004071, Homo sapiens CDC-like kinase 1 (CLK1).

(Target Sequences)

NM_(—)004071-1215,ccaggaaacgtaaatattt (SEQ ID NO: 139)

NM_(—)004071-774,catttcgactggatcatat (SEQ ID NO: 140)

NM_(—)004071-1216,caggaaacgtaaatatttt (SEQ ID NO: 141)

NM_(—)004071-973,ctttggtagtgcaacatat (SEQ ID NO: 142)

NM_(—)004071-463,cgtactaagtgcaagatat (SEQ ID NO: 143)

(Target Gene of RNAi)

NM_(—)001291, Homo sapiens CDC-like kinase 2 (CLK2).

(Target Sequences)

NM_(—)001291-202,gtatgaccggcgatactgt (SEQ ID NO: 144)

NM_(—)001291-225,gctacagacgcaacgatta (SEQ ID NO: 145)

NM_(—)001291-226,ctacagacgcaacgattat (SEQ ID NO: 146)

NM_(—)001291-45,ggagttaccgtgaacacta (SEQ ID NO: 147)

NM_(—)001291-46,gagttaccgtgaacactat (SEQ ID NO: 148)

(Target Gene of RNAi)

NM_(—)001292, Homo sapiens CDC-like kinase 3 (CLK3).

(Target Sequences)

NM_(—)001292-189,gccgtgacagcgatacata (SEQ ID NO: 149)

NM_(—)001292-72,cctacagtcgggaacatga (SEQ ID NO: 150)

NM_(—)001292-73,ctacagtcgggaacatgaa (SEQ ID NO: 151)

NM_(—)001292-188,cgccgtgacagcgatacat (SEQ ID NO: 152)

NM_(—)001292-121,gcctcccccacgaagatct (SEQ ID NO: 153)

(Target Sequence Effective for Mouse Homolog)

NM_(—)001292-388,ggtgaaggcacctttggca (SEQ ID NO: 154)

(Target Gene of RNAi)

NM_(—)020666, Homo sapiens CDC-like kinase 4 (CLK4).

(Target Sequences)

NM_(—)020666-617,gtattagagcacttaaata (SEQ ID NO: 155)

NM_(—)020666-1212,gaaaacgcaagtattttca (SEQ ID NO: 156)

NM_(—)020666-1348,cctggttcgaagaatgtta (SEQ ID NO: 157)

NM_(—)020666-181,cttgaatgagcgagattat (SEQ ID NO: 158)

NM_(—)020666-803,cagatctgccagtcaataa (SEQ ID NO: 159)

(Target Sequences Effective for Mouse Homolog)

NM_(—)020666-457,cgttctaagagcaagatat (SEQ ID NO: 160)

NM_(—)020.666-446,caaagtggagacgttctaa (SEQ ID NO: 161)

NM_(—)020666-461,ctaagagcaagatatgaaa (SEQ ID NO: 162)

(Target Gene of RNAi)

NM_(—)002093, Homo sapiens glycogen synthase kinase 3 beta (GSK3B).

(Target Sequences)

NM_(—)002093-326,gtccgattgcgttatttct (SEQ ID NO: 163)

NM_(—)002093-307,gctagatcactgtaacata (SEQ ID NO: 164)

NM_(—)002093-451,gacgctccctgtgatttat (SEQ ID NO: 165)

NM_(—)002093-632,cccaatgtttcgtatatct (SEQ ID NO: 166)

NM_(—)002093-623,cgaggagaacccaatgttt (SEQ ID NO: 167)

(Target Sequences Effective for Mouse Homolog)

NM_(—)002093-206,gtatatcaagccaaacttt (SEQ ID NO: 168)

NM_(—)002093-195,catttggtgtggtatatca (SEQ ID NO: 169)

NM_(—)002093-205,ggtatatcaagccaaactt (SEQ ID NO: 170)

(Target Gene of RNAi)

NM_(—)182691, Homo sapiens SFRS protein kinase 2 (SRPK2).

(Target Sequences)

NM_(—)182691-1312,gccaaatggacgacataaa (SEQ ID NO: 171)

NM_(—)182691-1313,ccaaatggacgacataaaa (SEQ ID NO: 172)

NM_(—)182691-1314,caaatggacgacataaaat (SEQ ID NO: 173)

NM_(—)182691-1985,ctgatcccgatgttagaaa (SEQ ID NO: 174)

NM_(—)182691-233,ggccggtatcatgttatta (SEQ ID NO: 175)

(Target Gene of RNAi)

NM_(—)005430, Homo sapiens wingless-type MMTV integration site family,member 1 (WNT1).

(Target Sequences)

NM_(—)005430-614,ggccgtacgaccgtattct (SEQ ID NO: 176)

NM_(—)005430-205,gcgtctgatacgccaaaat (SEQ ID NO: 177)

NM_(—)005430-855,cccacgacctcgtctactt (SEQ ID NO: 178)

NM_(—)005430-196,caaacagcggcgtctgata (SEQ ID NO: 179)

(Target Sequences Effective for Mouse Homolog)

NM_(—)005430-875,gagaaatcgcccaacttct (SEQ ID NO: 180)

NM_(—)005430-863,ctcgtctacttcgagaaat (SEQ ID NO: 181)

NM_(—)005430-860,gacctcgtctacttcgaga (SEQ ID NO: 182)

(Target Gene of RNAi)

NM_(—)003391, Homo sapiens wingless-type MMTV integration site familymember 2 (WNT2).

(Target Sequences)

NM_(—)003391-111,gggtgatgtgcgataatgt (SEQ ID NO: 183)

NM_(—)003391-681,ggaaaacgggcgattatct (SEQ ID NO: 184)

NM_(—)003391-764,gctaacgagaggtttaaga (SEQ ID NO: 185)

NM_(—)003391-765,ctaacgagaggtttaagaa (SEQ ID NO: 186)

NM_(—)003391-295,ggtcctactccgaagtagt (SEQ ID NO: 187)

(Target Sequences Effective for Mouse Homolog)

NM_(—)003391-797,gacctcgtgtattttgaga (SEQ ID NO: 188)

NM_(—)003391-790,gaaaaatgacctcgtgtat (SEQ ID NO: 189)

NM_(—)003391-789,cgaaaaatgacctcgtgta (SEQ ID NO: 190)

(Target Gene of RNAi)

NM_(—)004625, Homo sapiens wingless-type MMTV integration site family,member 7A (WNT7A).

(Target Sequences)

NM_(—)004625-92,ctgggcgcaagcatcatct (SEQ ID NO: 191)

NM_(—)004625-313,gttcacctacgccatcatt (SEQ ID NO: 192)

NM_(—)004625-524,gcccggactctcatgaact (SEQ ID NO: 193)

NM_(—)004625-480,gcttcgccaaggtctttgt (SEQ ID NO: 194)

(Target Sequences effective for mouse homolog)

NM_(—)004625-205,cctggacgagtgtcagttt (SEQ ID NO: 195)

NM_(—)004625-209,gacgagtgtcagtttcagt (SEQ ID NO: 196)

NM_(—)004625-172,catcatcgtcataggagaa (SEQ ID NO: 197)

(Target Gene of RNAi)

NM_(—)004626, Homo sapiens wingless-type MMTV integration site family,member 11 (WNT7A).

(Target Sequences)

NM_(—)004626-543,gatcccaagccaataaact (SEQ ID NO: 198)

NM_(—)004626-917,gacagctgcgaccttatgt (SEQ ID NO: 199)

NM_(—)004626-915,gcgacagctgcgaccttat (SEQ ID NO: 200)

NM_(—)004626-54,ccggcgtgtgctatggcat (SEQ ID NO: 201)

(Target Sequences Effective for Mouse Homolog)

NM_(—)004626-59,gtgtgctatggcatcaagt (SEQ ID NO: 202)

NM_(—)004626-560,ctgatgcgtctacacaaca (SEQ ID NO: 203)

NM_(—)004626-562,gatgcgtctacacaacagt (SEQ ID NO: 204)

(Target Gene of RNAi)

NM_(—)030753, Homo sapiens wingless-type MMTV integration site family,member 3 (WNT3).

(Target Sequences)

NM_(—)030753-417,gctgtgactcgcatcataa (SEQ ID NO: 205)

NM_(—)030753-483,ctgacttcggcgtgttagt (SEQ ID NO: 206)

NM_(—)030753-485,gacttcggcgtgttagtgt (SEQ ID NO: 207)

(Target Sequences Effective for Mouse Homolog)

NM_(—)030753-887,gaccggacttgcaatgtca (SEQ ID NO: 208)

NM_(—)030753-56,ctcgctggctacccaattt (SEQ ID NO: 209)

NM_(—)030753-59,gctggctacccaatttggt (SEQ ID NO: 210)

(Target Gene of RNAi)

NM_(—)033131, Homo sapiens wingless-type MMTV integration site family,member 3A (WNT3A).

(Target Sequences)

NM_(—)033131-2,gccccactcggatacttct (SEQ ID NO: 211)

NM_(—)033131-3,ccccactcggatacttctt (SEQ ID NO: 212)

NM_(—)033131-4,cccactcggatacttctta (SEQ ID NO: 213)

NM_(—)033131-77,gctgttgggccacagtatt (SEQ ID NO: 214)

NM_(—)033131-821,gaggcctcgcccaacttct (SEQ ID NO: 215)

(Target Sequences Effective for Mouse Homolog)

NM_(—)033131-168,ggaactacgtggagatcat (SEQ ID NO: 216)

NM_(—)033131-50,ggcagctacccgatctggt (SEQ ID NO: 217)

NM_(—)033131-165,gcaggaactacgtggagat (SEQ ID NO: 218)

(Target Gene of RNAi)

NM_(—)003392, Homo sapiens wingless-type MMTV integration site family,member 5A (WNT5A).

(Target Sequences)

NM_(—)003392-91,gtggtcgctaggtatgaat (SEQ ID NO: 219)

NM_(—)003392-93,ggtcgctaggtatgaataa (SEQ ID NO: 220)

NM_(—)003392-307,ggataacacctctgttttt (SEQ ID NO: 221)

NM_(—)003392-57,ccttcgcccaggttgtaat (SEQ ID NO: 222)

NM_(—)003392-87,cttggtggtcgctaggtat (SEQ ID NO: 223)

(Target Sequences Effective for Mouse Homolog)

NM_(—)003392-163,ccaactggcaggactttct (SEQ ID NO: 224)

NM_(—)003392-116,gttcagatgtcagaagtat (SEQ ID NO: 225)

NM_(—)003392-102,gtatgaataaccctgttca (SEQ ID NO: 226)

(Target Gene of RNAi)

NM_(—)004196, Homo sapiens cyclin-dependent kinase-like 1 (CDC2-relatedkinase) (CDKL1).

(Target Sequences)

NM_(—)004196-405,cgaaacattccgtgattaa (SEQ ID NO: 227)

NM_(—)004196-305,ctcgtgaagagcataactt (SEQ ID NO: 228)

NM_(—)004196-458,ggaccgagtgactactata (SEQ ID NO: 229)

NM_(—)004196-844,gttgcatcacccatatttt (SEQ ID NO: 230)

NM_(—)004196-330,cactgcaagctgtaaattt (SEQ ID NO: 231)

(Target Sequence Effective for Mouse Homolog)

NM_(—)004196-119,gatgaccctgtcataaaga (SEQ ID NO: 232)

(Target Gene of RNAi)

NM_(—)003948, Homo sapiens cyclin-dependent kinase-like 2 (CDC2-relatedkinase) (CDKL2).

(Target Sequences)

NM_(—)003948-623,gatcagctatatcatatta (SEQ ID NO: 233)

NM_(—)003948-1379,ccatcaggcatttataaca (SEQ ID NO: 234)

NM_(—)003948-1380,catcaggcatttataacat (SEQ ID NO: 235)

NM_(—)003948-768,ctgaagtggtgatagattt (SEQ ID NO: 236)

NM_(—)003948-626,cagctatatcatattatga (SEQ ID NO: 237)

(Target Sequences Effective for Mouse Homolog)

NM_(—)003948-325,gattattaatggaattgga (SEQ ID NO: 238)

NM_(—)003948-1012,ggtacaggataccaatgct (SEQ ID NO: 239)

(Target Gene of RNAi)

NM_(—)016508, Homo sapiens cyclin-dependent kinase-like 3 (CDKL3).

(Target Sequences)

NM_(—)016508-498,gagctcccgaattagtatt (SEQ ID NO: 240)

NM_(—)016508-500,gctcccgaattagtattaa (SEQ ID NO: 241)

NM_(—)016508-1290,cacccatcaatctaactaa (SEQ ID NO: 242)

NM_(—)016508-1301,ctaactaacagtaatttga (SEQ ID NO: 243)

NM_(—)016508-501,ctcccgaattagtattaaa (SEQ ID NO: 244)

(Target Sequences Effective for Mouse Homolog)

NM_(—)016508-785,gttcatgcttgtttacaaa (SEQ ID NO: 245)

NM_(—)016508-555,ctttgggctgtatgatcat (SEQ ID NO: 246)

NM_(—)016508-776,gcagatatagttcatgctt (SEQ ID NO: 247).

(Target Gene of RNAi)

NM_(—)002745, Homo sapiens mitogen-activated protein kinase 1 (MAPK1).

(Target Sequences)

NM_(—)002745-746,gaagacctgaattgtataa (SEQ ID NO: 248)

NM_(—)002745-276,caaccatcgagcaaatgaa (SEQ ID NO: 249)

NM_(—)002745-849,ccaaagctctggacttatt (SEQ ID NO: 250)

NM_(—)002745-749,gacctgaattgtataataa (SEQ ID NO: 251)

NM_(—)002745-113,gtgtgctctgcttatgata (SEQ ID NO: 252)

(Target Sequences Effective for Mouse Homolog)

NM_(—)002745-220,cttactgcgcttcagacat (SEQ ID NO: 253)

NM_(—)002745-228,gcttcagacatgagaacat (SEQ ID NO: 254)

NM_(—)002745-224,ctgcgcttcagacatgaga (SEQ ID NO: 255)

(Target Gene of RNAi)

NM_(—)016231, Homo sapiens nemo-like kinase (NLK).

(Target Sequences)

NM_(—)016231-450,gagtagcgctcaaaaagat (SEQ ID NO: 256)

NM_(—)016231-1074,gcgctaaggcacatatact (SEQ ID NO: 257)

NM_(—)016231-962,ctactaggacgaagaatat (SEQ ID NO: 258)

NM_(—)016231-579,ctccacacattgactattt (SEQ ID NO: 259)

(Target Sequences Effective for Mouse Homolog)

NM_(—)016231-703,gattttgcgaggtttgaaa (SEQ ID NO: 260)

NM_(—)016231-1382,gtccgacaggttaaagaaa (SEQ ID NO: 261)

NM_(—)016231-1384,ccgacaggttaaagaaatt (SEQ ID NO: 262)

(Target Gene of RNAi)

NM_(—)001315, Homo sapiens mitogen-activated protein kinase 14 (MAPK14).

(Target Sequences)

NM_(—)001315-401,ctccgaggtctaaagtata (SEQ ID NO: 263)

NM_(—)001315-403,ccgaggtctaaagtatata (SEQ ID NO: 264)

NM_(—)001315-251,ggtctgttggacgttttta (SEQ ID NO: 265)

NM_(—)001315-212,ctgcggttacttaaacata (SEQ ID NO: 266)

NM_(—)001315-405,gaggtctaaagtatataca (SEQ ID NO: 267)

(Target Sequence Effective for Mouse Homolog)

NM_(—)001315-664,gtttcctggtacagaccat (SEQ ID NO: 268)

(Target Gene of RNAi)

NM_(—)002751, Homo sapiens mitogen-activated protein kinase 11 (MAPK11).

(Target Sequences)

NM_(—)002751-366,gcgacgagcacgttcaatt (SEQ ID NO: 269)

NM_(—)002751-667,cccgggaagcgactacatt (SEQ ID NO: 270)

NM_(—)002751-669,cgggaagcgactacattga (SEQ ID NO: 271)

NM_(—)002751-731,gaggttctggcaaaaatct (SEQ ID NO: 272)

NM_(—)002751-729,ctgaggttctggcaaaaat (SEQ ID NO: 273)

(Target Gene of RNAi)

NM_(—)002969, Homo sapiens mitogen-activated protein kinase 12 (MAPK12).

(Target Sequences)

NM_(—)002969-1018,gaagcgtgttacttacaaa (SEQ ID NO: 274)

NM_(—)002969-262,gctgctggacgtattcact (SEQ ID NO: 275)

NM_(—)002969-1017,ggaagcgtgttacttacaa (SEQ ID NO: 276)

NM_(—)002969-578,cccgaggtcatcttgaatt (SEQ ID NO: 277)

NM_(—)002969-1013,gaatggaagcgtgttactt (SEQ ID NO: 278)

(Target Gene of RNAi)

NM_(—)002754, Homo sapiens mitogen-activated protein kinase 13 (MAPK13).

(Target Sequences)

NM_(—)002754-164,ctgagccgaccctttcagt (SEQ ID NO: 279)

NM_(—)002754-174,cctttcagtccgagatctt (SEQ ID NO: 280)

NM_(—)002754-978,ccttagaacacgagaaact (SEQ ID NO: 281)

NM_(—)002754-285,ccctgcgcaacttctatga (SEQ ID NO: 282)

NM_(—)002754-287,ctgcgcaacttctatgact (SEQ ID NO: 283)

(Target Gene of RNAi)

NM_(—)139049, Homo sapiens mitogen-activated protein kinase 8 (MAPK8).

(Target Sequences)

NM_(—)139049-449,gacttaaagcccagtaata (SEQ ID NO: 284)

NM_(—)139049-213,gagagctagttcttatgaa (SEQ ID NO: 285)

NM_(—)139049-451,cttaaagcccagtaatata (SEQ ID NO: 286)

(Target Sequences Effective for Mouse Homolog)

NM_(—)139049-525,caggaacgagttttatgat (SEQ ID NO: 287)

NM_(—)139049-524,gcaggaacgagttttatga (SEQ ID NO: 288)

NM_(—)139049-283,gaaatccctagaagaattt (SEQ ID NO: 289)

(Target Gene of RNAi)

NM_(—)002752, Homo sapiens mitogen-activated protein kinase 9 (MAPK9).

(Target Sequences)

NM_(—)002752-116,gtttgtgctgcatttgata (SEQ ID NO: 290)

NM_(—)002752-204,gagcttatcgtgaacttgt (SEQ ID NO: 291)

(Target Sequences Effective for Mouse Homolog)

NM_(—)002752-878,gccagagatctgttatcaa (SEQ ID NO: 292)

NM_(—)002752-879,ccagagatctgttatcaaa (SEQ ID NO: 293)

NM_(—)002752-880,cagagatctgttatcaaaa (SEQ ID NO: 294)

(Target Gene of RNAi)

NM_(—)002753, Homo sapiens mitogen-activated protein kinase 10 (MAPK10).

(Target Sequences)

NM_(—)002753-668,gtggtgacacgttattaca (SEQ ID NO: 295)

NM_(—)002753-957,cggactccgagcacaataa (SEQ ID NO: 296)

NM_(—)002753-958,ggactccgagcacaataaa (SEQ ID NO: 297)

NM_(—)002753-811,gtggaataaggtaattgaa (SEQ ID NO: 298)

NM_(—)002753-1212,ctaaaaatggtgtagtaaa (SEQ ID NO: 299)

(Target Sequences Effective for Mouse Homolog)

NM_(—)002753-1167,ggaaagaacttatctacaa (SEQ ID NO: 300)

NM_(—)002753-584,gtagtcaagtctgattgca (SEQ ID NO: 301)

NM_(—)002753-761,gaaatggttcgccacaaaa (SEQ ID NO: 302)

(Target Gene of RNAi)

NM_(—)001786, Homo sapiens cell division cycle 2, G1 to S and G2 to M(CDC2).

(Target Sequences)

NM_(—)001786-782,gatttgctctcgaaaatgt (SEQ ID NO: 303)

NM_(—)001786-788,ctctcgaaaatgttaatct (SEQ ID NO: 304)

NM_(—)001786-658,gggcactcccaataatgaa (SEQ ID NO: 305)

NM_(—)001786-696,ctttacaggactataagaa (SEQ ID NO: 306)

NM_(—)001786-562,gagtataggcaccatattt (SEQ ID NO: 307)

(Target Sequence Effective for Mouse Homolog)

NM_(—)001786-869,gacaatcagattaagaaga (SEQ ID NO: 308)

(Target Gene of RNAi)

NM_(—)001798, Homo sapiens cyclin-dependent kinase 2 (CDK2).

(Target Sequences)

NM_(—)001798-224,ctctacctggtttttgaat (SEQ ID NO: 309)

NM_(—)001798-690,cttctatgcctgattacaa (SEQ ID NO: 310)

NM_(—)001798-770,gatggacggagcttgttat (SEQ ID NO: 311)

NM_(—)001798-226,ctacctggtttttgaattt (SEQ ID NO: 312)

NM_(—)001798-36,gcacgtacggagttgtgta (SEQ ID NO: 313)

(Target Gene of RNAi)

NM_(—)000075, Homo sapiens cyclin-dependent kinase 4 (CDK4).

(Target Sequences)

NM_(—)000075-45,cctatgggacagtgtacaa (SEQ ID NO: 314)

NM_(—)000075-616,gatgtttcgtcgaaagcct (SEQ ID NO: 315)

NM_(—)000075-161,cgtgaggtggctttactga (SEQ ID NO: 316)

NM_(—)000075-35,ggtgtcggtgcctatggga (SEQ ID NO: 317)

NM_(—)000075-242,cgaactgaccgggagatca (SEQ ID NO: 318)

(Target Gene of RNAi)

NM_(—)052984, Homo sapiens cyclin-dependent kinase 4 (CDK4), transcriptvariant 2, mRNA.,228 . . . 563,0

(Target Sequences)

NM_(—)052984-248,gaccgggagatcaagagat (SEQ ID NO: 319)

NM_(—)052984-251,cgggagatcaagagatgtt (SEQ ID NO: 320)

(Target Gene of RNAi)

NM_(—)001799, Homo sapiens cyclin-dependent kinase 7 (MO15 homolog,Xenopus laevis, cdk-activating kinase) (CDK7).

(Target Sequences)

NM_(—)001799-242,ggacataaatctaatatta (SEQ ID NO: 321)

NM_(—)001799-104,caaattgtcgccattaaga (SEQ ID NO: 322)

NM_(—)001799-490,ccccaatagagcttataca (SEQ ID NO: 323)

NM_(—)001799-20,cgggcaaagcgttatgaga (SEQ ID NO: 324)

NM_(—)001799-21,gggcaaagcgttatgagaa (SEQ ID NO: 325)

(Target Sequence Effective for Mouse Homolog)

NM_(—)001799-345,cctacatgttgatgactct (SEQ ID NO: 326)

(Target Gene of RNAi)

NM_(—)000455, Homo sapiens serine/threonine kinase 11 (Peutz-Jegherssyndrome) (STK11).

(Target Sequences)

NM_(—)000455-306,ggaggttacggcacaaaaa (SEQ ID NO: 327)

NM_(—)000455-307,gaggttacggcacaaaaat (SEQ ID NO: 328)

NM_(—)000455-309,ggttacggcacaaaaatgt (SEQ ID NO: 329)

NM_(—)000455-1157,cccaaggccgtgtgtatga (SEQ ID NO: 330)

NM_(—)000455-1158,ccaaggccgtgtgtatgaa (SEQ ID NO: 331)

(Target Sequence Effective for Mouse Homolog)

NM_(—)000455-916,cagctggttccggaagaaa (SEQ ID NO: 332)

(Target Gene of RNAi)

NM_(—)001274, Homo sapiens CHK1 checkpoint homolog (S. pombe) (CHEK1).

(Target Sequences)

NM_(—)001274-456,cagtatttcggtataataa (SEQ ID NO: 333)

NM_(—)001274-361,gcatggtattggaataact (SEQ ID NO: 334)

NM_(—)001274-990,gcccctcatacattgataa (SEQ ID NO: 335)

NM_(—)001274-1038,ccacatgtcctgatcatat (SEQ ID NO: 336)

NM_(—)001274-227,ggcaatatccaatatttat (SEQ ID NO: 337)

(Target Sequences Effective for Mouse Homolog)

NM_(—)001274-573,ggtcctgtggaatagtact (SEQ ID NO: 338)

NM_(—)001274-416,gaaagggataacctcaaaa (SEQ ID NO: 339)

NM_(—)001274-577,ctgtggaatagtacttact (SEQ ID NO: 340)

(Target Gene of RNAi)

NM_(—)002648, Homo sapiens pim-1 oncogene (PIM1).

(Target Sequences)

NM_(—)002648-831,ggccaaccttcgaagaaat (SEQ ID NO: 341)

NM_(—)002648-601,cgatgggacccgagtgtat (SEQ ID NO: 342)

NM_(—)002648-602,gatgggacccgagtgtata (SEQ ID NO: 343)

NM_(—)002648-293,ggtttctccggcgtcatta (SEQ ID NO: 344)

NM_(—)002648-834,caaccttcgaagaaatcca (SEQ ID NO: 345)

(Target Sequences Effective for Mouse Homolog)

NM_(—)002648-96-ccctggagtcgcagtacca (SEQ ID NO: 346)

NM_(—)002648-203,gtggagaaggaccggattt (SEQ ID NO: 347)

(Target Gene of RNAi)

NM_(—)006875, Homo sapiens pim-2 oncogene (PIM2).

(Target Sequences)

NM_(—)006875-698,ggggacattccctttgaga (SEQ ID NO: 348)

NM_(—)006875-242,ctcgaagtcgcactgctat (SEQ ID NO: 349)

NM_(—)006875-245,gaagtcgcactgctatgga (SEQ ID NO: 350)

NM_(—)006875-499,gaacatcctgatagaccta (SEQ ID NO: 351)

NM_(—)006875-468,gtggagttgtccatcgtga (SEQ ID NO: 352)

(Target Gene of RNAi)

NM_(—)021643, Homo sapiens tribbles homolog 2 (TRB2).

(Target Sequences)

NM_(—)021643-174,cttgtatcgggaaatactt (SEQ ID NO: 353)

NM_(—)021643-71,gaagagttgtcgtctataa (SEQ ID NO: 354)

NM_(—)021643-177,gtatcgggaaatacttatt (SEQ ID NO: 355)

NM_(—)021643-524,ctcaagctgcggaaattca (SEQ ID NO: 356)

(Target Sequences Effective for Mouse Homolog)

NM_(—)021643-41,gggagatcgcggaacaaaa (SEQ ID NO: 357)

NM_(—)021643-382,gttctttgagcgaagctat (SEQ ID NO: 358)

NM_(—)021643-143,cccgagactccgaacttgt (SEQ ID NO: 359)

(Target Gene of RNAi)

NM_(—)007118, Homo sapiens triple functional domain (PTPRF interacting)(TRIO).

(Target Sequences)

NM_(—)007118-1684,caccaatgcggataaatta (SEQ ID NO: 360)

NM_(—)007118-1686,ccaatgcggataaattact (SEQ ID NO: 361)

NM_(—)007118-3857,gaaatctacgaatttcata (SEQ ID NO: 362)

NM_(—)007118-6395,gagcagatcgtcatattca (SEQ ID NO: 363)

NM_(—)007118-8531,cctatccgtagcattaaaa (SEQ ID NO: 364)

(Target Gene of RNAi)

NM_(—)004938, Homo sapiens death-associated protein kinase 1 (DAPK1).

(Target Sequences)

NM_(—)004938-917,caatccgttcgcttgatat (SEQ ID NO: 365)

NM_(—)004938-1701,ggtgtttcgtcgattatca (SEQ ID NO: 366)

NM_(—)004938-1702,gtgtttcgtcgattatcaa (SEQ ID NO: 367)

NM_(—)004938-2824,gaaggtacttcgaaatcat (SEQ ID NO:.368)

NM_(—)004938-668,gaaacgttagcaaatgtat (SEQ ID NO: 369)

(Target Sequences Effective for Mouse Homolog)

NM_(—)004938-609,gggtaataacctatatcct (SEQ ID NO: 370)

NM_(—)004938-2697,gaggcgagtttggatatga (SEQ ID NO: 371)

NM_(—)004938-490,ggcccataaaattgacttt (SEQ ID NO: 372)

(Target Gene of RNAi)

NM_(—)006252, Homo sapiens protein kinase, AMP-activated, alpha 2catalytic subunit (PRKAA2).

(Target Sequences)

NM_(—)006252-760,gaaacgagcaactatcaaa (SEQ ID NO: 373)

NM_(—)006252-148,gaagattcgcagtttagat (SEQ ID NO: 374)

NM_(—)006252-1227,gcaaaccgtatgacattat (SEQ ID NO: 375)

NM_(—)006252-1338,ctggcaattacgtgaaaat (SEQ ID NO: 376)

NM_(—)006252-1340,ggcaattacgtgaaaatga (SEQ ID NO: 377)

(Target Gene of RNAi)

NM_(—)002742, Homo sapiens protein kinase C, mu (PRKCM).

(Target Sequences)

NM_(—)002742-508,ggtacgtcaaggtcttaaa (SEQ ID NO: 378)

NM_(—)002742-1332,gattggatagcaaatgtat (SEQ ID NO: 379)

NM_(—)002742-509,gtacgtcaaggtcttaaat (SEQ ID NO: 380)

NM_(—)002742-370,ggaaggcgatcttattgaa (SEQ ID NO: 381)

(Target Sequences Effective for Mouse Homolog)

NM_(—)002742-1913,caccctggtgttgtaaatt (SEQ ID NO: 382)

NM_(—)002742-2041,cataacgaagtttttaatt (SEQ ID NO: 383)

NM_(—)002742-2521,ctatcagacctggttagat (SEQ ID NO: 384)

(Target Gene of RNAi)

NM 003684, Homo sapiens MAP kinase-interacting serine/threonine kinase 1(MKNK1).

(Target Sequences)

NM_(—)003684-218,gagtatgccgtcaaaatca (SEQ ID NO: 385)

NM_(—)003684-229,caaaatcatcgagaaacaa (SEQ ID NO: 386)

NM_(—)003684-344,gatgacacaaggttttact (SEQ ID NO: 387)

NM_(—)003684-192,gtgccgtgagcctacagaa (SEQ ID NO: 388)

NM_(—)003684-379,gcaaggaggttccatctta (SEQ ID NO: 389)

(Target Gene of RNAi)

NM_(—)004759, Homo sapiens mitogen-activated protein kinase-activatedprotein kinase 2 (MAPKAPK2).

(Target Sequences)

NM_(—)004759-942,ccatcaccgagtttatgaa (SEQ ID NO: 390)

NM_(—)004759-836,cgaatgggccagtatgaat (SEQ ID NO: 391)

NM_(—)004759-563,cctgagaatctcttataca (SEQ ID NO: 392)

NM_(—)004759-669,gttatacaccgtactatgt (SEQ ID NO: 393)

NM_(—)004759-362,gatgtgtacgagaatctgt (SEQ ID NO: 394)

(Target Gene of RNAi)

NM_(—)172171, Homo sapiens calcium/calmodulin-dependent protein kinase(CaM kinase) II gamma (CAMK2G).

(Target Sequences)

NM_(—)172171-113,gagtacgcagcaaaaatca (SEQ ID NO: 395)

NM_(—)172171-422,ctgctgctggcgagtaaat (SEQ ID NO: 396)

NM_(—)172171-1075,ggtacacaacgctacagat (SEQ ID NO: 397)

NM_(—)172171-474,gcctagccatcgaagtaca (SEQ ID NO: 398)

(Target Sequences Effective for Mouse Homolog)

NM_(—)172171-425,ctgctggcgagtaaatgca (SEQ ID NO: 399)

NM_(—)172171-260,ctcgtgtttgaccttgtta (SEQ ID NO: 400)

NM_(—)172171-597,gcggggtcatcctgtatat (SEQ ID NO: 401)

(Target Gene of RNAi)

NM_(—)015981, Homo sapiens calcium/calmodulin-dependent protein kinase(CaM kinase) II alpha (CAMK2A).

(Target Sequences)

NM_(—)015981-1213,ccatcgattctattttgaa (SEQ ID NO: 402)

NM_(—)015981-1210,cttccatcgattctatttt (SEQ ID NO: 403)

NM_(—)015981-1067,cggaaacaggaaattataa (SEQ ID NO: 404)

NM_(—)015981-1066,gcggaaacaggaaattata (SEQ ID NO: 405)

NM_(—)015981-754,gaccattaacccatccaaa (SEQ ID NO: 406)

(Target Sequences Effective for Mouse Homolog)

NM_(—)015981-1130,gagtcctacacgaagatgt (SEQ ID NO: 407)

NM_(—)015981-1416,ggcagatcgtccacttcca (SEQ ID NO: 408)

NM_(—)015981-1418,cagatcgtccacttccaca (SEQ ID NO: 409)

(Target Gene of RNAi)

NM_(—)020439, Homo sapiens calcium/calmodulin-dependent protein kinaseIG (CAMK1G).

(Target Sequences)

NM_(—)020439-1354,ggtcatggtaccagttaaa (SEQ ID NO: 410)

NM_(—)020439-1409,ggagtctgtctcattatgt (SEQ ID NO: 411)

NM_(—)020439-639,gtggataccccccattcta (SEQ ID NO: 412)

NM_(—)020439-823,ctggattgacggaaacaca (SEQ ID NO: 413)

NM_(—)020439-662,gaaacggagtctaagcttt (SEQ ID NO: 414)

(Target Sequences Effective for Mouse Homolog)

NM_(—)020439-85,gggatcaggagctttctca (SEQ ID NO: 415)

NM_(—)020439-903,gcaagtggaggcaagcctt (SEQ ID NO: 416)

(Target Gene of RNAi)

NM_(—)007194, Homo sapiens CHK2 checkpoint homolog (S. pombe) (CHEK2).

(Target Sequences)

NM_(—)007194-460,ctcttacattgcatacata (SEQ ID NO: 417)

NM_(—)007194-201,ctcaggaactctattctat (SEQ ID NO: 418)

NM_(—)007194-1233,gtttaggagttattctttt (SEQ ID NO: 419)

NM_(—)007194-398,gataaataccgaacataca (SEQ ID NO: 420)

NM_(—)007194-396,cagataaataccgaacata (SEQ ID NO: 421)

(Target Sequences Effective for Mouse Homolog)

NM_(—)007194-614,gtagatgatcagtcagttt (SEQ ID NO: 422)

NM_(—)007194-620,gatcagtcagtttatccta (SEQ ID NO: 423)

NM_(—)007194-612,ctgtagatgatcagtcagt (SEQ ID NO: 424)

(Target Gene of RNAi)

NM_(—)002610, Homo sapiens pyruvate dehydrogenase kinase, isoenzyme 1(PDK1).

(Target Sequences)

NM_(—)002610-1194,gactcccagtgtataacaa (SEQ ID NO: 425)

NM_(—)002610-553,catgagtcgcatttcaatt (SEQ ID NO: 426)

NM_(—)002610-306,-ggacaccatccgttcaatt (SEQ ID NO: 427)

NM_(—)002610-1086,gtctttacgcacaatactt (SEQ ID NO: 428)

NM_(—)002610-388,ggatgctaaagctatttat (SEQ ID NO: 429)

(Target Gene of RNAi)

NM_(—)001619, Homo sapiens adrenergic, beta, receptor kinase 1 (ADRBK1)

(Target Sequences)

NM_(—)001619-474,gggacgtgttccagaaatt (SEQ ID NO: 430)

NM_(—)001619-317,gagatcttcgactcataca (SEQ ID NO: 431)

NM_(—)001619-665,gacaaaaagcgcatcaaga (SEQ ID NO: 432)

NM_(—)001619-439,gccatacatcgaagagatt (SEQ ID NO: 433)

NM_(—)001619-476,gacgtgttccagaaattca (SEQ ID NO: 434)

(Target Sequences Effective for Mouse Homolog)

NM_(—)001619-1476,caaaaggaatcaagttact (SEQ ID NO: 435)

NM_(—)001619-1474,cacaaaaggaatcaagtta (SEQ ID NO: 436)

NM_(—)001619-1171,ccggcagcacaagaccaaa (SEQ ID NO: 437)

(Target Gene of RNAi)

NM_(—)005160, Homo sapiens adrenergic, beta, receptor kinase 2 (ADRBK2).

(Target Sequences)

NM_(—)005160-1779,gagagtcccggcaaaattt (SEQ ID NO: 438)

NM_(—)005160-1778,ggagagtcccggcaaaatt (SEQ ID NO: 439)

NM_(—)005160-1373,cagcatgtctacttacaaa (SEQ ID NO: 440)

NM_(—)005160-307,cagaagtcgacaaatttat (SEQ ID NO: 441)

NM_(—)005160-306,gcagaagtcgacaaattta (SEQ ID NO: 442)

(Target Gene of RNAi)

NM_(—)003161, Homo sapiens ribosomal protein S6 kinase, 7OkDa,polypeptide 1 (RPS6KB1).

(Target Sequences)

NM_(—)003161-1294,ccgatcacctcgaagattt (SEQ ID NO: 443)

NM_(—)003161-1556,cacctgcgtatgaatctat (SEQ ID NO: 444)

NM_(—)003161-1296,gatcacctcgaagatttat (SEQ ID NO: 445)

NM_(—)003161-831,gtttgggagcattaatgta (SEQ ID NO: 446)

NM_(—)003161-1295,cgatcacctcgaagattta (SEQ ID NO: 447)

(Target Gene of RNAi)

NM_(—)014496, Homo sapiens ribosomal protein S6 kinase, 9OkDa,polypeptide 6 (RPS6KA6).

(Target Sequences)

NM_(—)014496-682,gaaggcttactcattttgt (SEQ ID NO: 448)

NM_(—)014496-1552,ggaggctagtgatatacta (SEQ ID NO: 449)

NM_(—)014496-1553,gaggctagtgatatactat (SEQ ID NO: 450)

NM_(—)014496-1551,gggaggctagtgatatact (SEQ ID NO: 451)

NM_(—)014496-1481,cttgttacggatttaatga (SEQ ID NO: 452)

(Target Sequences Effective for Mouse Homolog)

NM_(—)014496-831,gaaatgagaccatgaatat (SEQ ID NO: 453)

NM_(—)014496-1411,gatgcgctatggacaacat (SEQ ID NO: 454)

NM_(—)014496-927,ggaatccagcaaatagatt (SEQ ID NO: 455)

(Target Gene of RNAi)

NM_(—)002953, Homo sapiens ribosomal protein S6 kinase, 9OkDa,polypeptide 1 (RPS6KA1).

(Target Sequences)

NM_(—)002953-739,ctatggggtgttgatgttt (SEQ ID NO: 456)

NM_(—)002953-1331,gctgtcaaggtcattgata (SEQ ID NO: 457)

NM_(—)002953-1332,ctgtcaaggtcattgataa (SEQ ID NO: 458)

NM_(—)002953-735,ggtcctatggggtgttgat (SEQ ID NO: 459)

NM_(—)002953-738,cctatggggtgttgatgtt (SEQ ID NO: 460)

(Target Sequences Effective for Mouse Homolog)

NM_(—)002953-666,gcgggacagtggagtacat (SEQ ID NO: 461)

NM_(—)002953-832,gctaggcatgccccagttt (SEQ ID NO: 462)

NM_(—)002953-1315,caccaacatggagtatgct (SEQ ID NO: 463)

(Target Gene of RNAi)

NM_(—)001626, Homo sapiens v-akt murine thymoma viral oncogene homolog 2(AKT2).

(Target Sequences)

NM_(—)001626-141,ctctaccccccttaaacaa (SEQ ID NO: 464)

NM_(—)001626-35,cacaagcgtggtgaataca (SEQ ID NO: 465)

NM_(—)001626-143,ctaccccccttaaacaact (SEQ ID NO: 466)

NM_(—)001626-41,cgtggtgaatacatcaaga (SEQ ID NO: 467)

NM_(—)001626-420,gcaaggcacgggctaaagt (SEQ ID NO: 468)

(Target Gene of RNAi)

NM_(—)005163, Homo sapiens v-akt murine thymoma viral oncogene homolog 1(AKT1).

(Target Sequences)

NM_(—)005163-1294,gactgacaccaggtatttt (SEQ ID NO: 469)

NM_(—)005163-1296,ctgacaccaggtattttga (SEQ ID NO: 470)

NM_(—)005163-1292,gagactgacaccaggtatt (SEQ ID NO: 471)

NM_(—)005163-751,cttctatggcgctgagatt (SEQ ID NO: 472)

NM_(—)005163-630,cagccctgaagtactcttt (SEQ ID NO: 473)

(Target Gene of RNAi)

NM_(—)005465, Homo sapiens v-akt murine thymoma viral oncogene homolog 3(protein kinase B, gamma) (AKT3).

(Target Sequences)

NM_(—)005465-229,ccagtggactactgttata (SEQ ID NO: 474)

NM_(—)005465-99,cattcataggatataaaga (SEQ ID NO: 475)

NM_(—)005465-402,cctctacaacccatcataa (SEQ ID NO: 476)

NM_(—)005465-1283,gagacagatactagatatt (SEQ ID NO: 477)

(Target Sequences Effective for Mouse Homolog)

NM_(—)005465-733,ggaccgcacacgtttctat (SEQ ID NO: 478)

NM_(—)005465-1317,cagctcagactattacaat (SEQ ID NO: 479)

NM_(—)005465-1319,gctcagactattacaataa (SEQ ID NO: 480)

(Target Gene of RNAi)

NM_(—)005627, Homo sapiens serum/glucocorticoid regulated kinase (SGK).

(Target Sequences)

NM_(—)005627-875,ggcctgccgcctttttata (SEQ ID NO: 481)

NM_(—)005627-97,gggtctgaacgactttatt (SEQ ID NO: 482)

NM_(—)005627-99,gtctgaacgactttattca (SEQ ID NO: 483)

NM_(—)005627-190,ggagcctgagcttatgaat (SEQ ID NO: 484)

NM_(—)005627-413,gaggagaagcatattatgt (SEQ ID NO: 485)

(Target Sequences Effective for Mouse Homolog)

NM_(—)005627-649,catcgtttatagagactta (SEQ ID NO: 486)

NM_(—)005627-367,ctatgcagtcaaagtttta (SEQ ID NO: 487)

NM_(—)005627-307,gatcggaaagggcagtttt (SEQ ID NO: 488)

(Target Gene of RNAi)

NM_(—)170693, Homo sapiens serum/glucocorticoid regulated kinase 2(SGK2).

(Target Sequences)

NM_(—)1,70693-163,gtctgatggggcgttctat (SEQ ID NO: 489)

NM_(—)170693-840,cagactttcttgagattaa (SEQ ID NO: 490)

NM_(—)170693-842,gactttcttgagattaaga (SEQ ID NO: 491)

NM_(—)170693-582,gtggtacccctgagtactt (SEQ ID NO: 492)

NM_(—)170693-183,cagtgaaggtactacagaa (SEQ ID′ NO: 493)

(Target Sequence Effective for Mouse Homolog)

NM_(—)170693-287,gtgggcctgcgctactcct (SEQ ID NO: 494)

(Target Gene of RNAi)

NM_(—)013257, Homo sapiens serum/glucocorticoid regulated kinase-like(SGKL).

(Target Sequences)

NM_(—)013257-273,caggactaaacgaattcat (SEQ ID NO: 495)

NM_(—)013257-944,gacaccactaccacatttt (SEQ ID NO: 496)

NM_(—)013257-1388,gtatcttctgactattcta (SEQ ID NO: 497)

NM_(—)013257-946,caccactaccacattttgt (SEQ ID NO: 498)

NM_(—)013257-790,gttttacgctgctgaaatt (SEQ ID NO: 499)

(Target Sequences Effective for Mouse Homolog)

NM_(—)013257-693,caactgaaaagctttattt (SEQ ID NO: 500)

NM_(—)013257-225,gaatatttggtgataattt (SEQ ID NO: 501)

NM_(—)013257-38,ccaagtgtaagcattccca (SEQ ID NO: 502)

(Target Gene of RNAi)

NM_(—)002744, Homo sapiens protein kinase C, zeta (PRKCZ).

(Target Sequences)

NM_(—)002744-1233,gcggaaccccgaattacat (SEQ ID NO: 503)

NM_(—)002744-398,caagccaagcgctttaaca (SEQ ID NO: 504)

NM_(—)002744-1447,caaagcctcccatgtttta (SEQ ID NO: 505)

NM_(—)002744-823,ccaaatttacgccatgaaa (SEQ ID NO: 506)

NM_(—)002744-1100,cacgagagggggatcatct (SEQ ID NO: 507)

(Target Gene of RNAi)

NM_(—)006254, Homo sapiens protein kinase C, delta (PRKCD).

(Target Sequences)

NM_(—)006254-1524,gcggcacccctgactatat (SEQ ID NO: 508)

NM_(—)006254-1339,ctaccgtgccacgttttat (SEQ ID NO: 509)

NM_(—)006254-992,gggacctacggcaagatct (SEQ ID NO: 510)

(Target Sequences Effective for Mouse Homolog)

NM_(—)006254-172,gttcgacgcccacatctat (SEQ ID NO: 511)

NM_(—)006254-659,cagaaagaacgcttcaaca (SEQ ID NO: 512)

NM_(—)006254-761,gtgaagcagggattaaagt (SEQ ID NO: 513)

(Target Gene of RNAi)

NM_(—)002737, Homo sapiens protein kinase C, alpha (PRKCA).

(Target Sequences)

NM_(—)002737-1571,ggcgtcctgttgtatgaaa (SEQ ID NO: 514)

NM_(—)002737-393,gtgacacctgcgatatgaa (SEQ ID NO: 515)

NM_(—)002737-711,gacgactgtctgtagaaat (SEQ ID NO: 516)

NM_(—)002737-1085,gaactgtatgcaatcaaaa (SEQ ID NO: 517)

NM_(—)002737-1924,gctggttattgctaacata (SEQ ID NO: 518)

(Target Sequences Effective for Mouse Homolog)

NM_(—)002737-1958,gaagggttctcgtatgtca (SEQ ID NO: 519)

NM_(—)002737-1835,ccattcaagcccaaagtgt (SEQ ID NO: 520)

NM_(—)002737-1234,gctgtacttcgtcatggaa (SEQ ID NO: 521)

(Target Gene of RNAi)

NM_(—)002738, Homo sapiens protein kinase C, beta 1 (PRKCB1).

(Target Sequences)

NM_(—)002738-573,cagatccctacgtaaaact (SEQ ID NO: 522)

NM_(—)002738-1791,catttttccggtatattga (SEQ ID NO: 523)

NM_(—)002738-1384,catttaccgtgacctaaaa (SEQ ID NO: 524)

NM_(—)002738-575,gatccctacgtaaaactga (SEQ ID NO: 525)

NM_(—)002738-1315,ggagccccatgctgtattt (SEQ ID NO: 526)

(Target Sequences Effective for Mouse Homolog)

NM_(—)002738-1006,gatgaaactgaccgatttt (SEQ ID NO: 527)

NM_(—)002738-1961,gaattcgaaggattttcct (SEQ ID NO: 528)

NM_(—)002738-1233,ccatggaccgcctgtactt (SEQ ID NO: 529)

(Target Gene of RNAi)

NM_(—)015282, Homo sapiens cytoplasmic linker associated protein 1(CLASP1).

(Target Sequences)

NM_(—)015282-2447,gagccgtatgggatgtatt (SEQ ID NO: 530)

NM_(—)015282-4151,gccgagctgacgattatga (SEQ ID NO: 531)

NM_(—)015282-4152,ccgagctgacgattatgaa (SEQ ID NO: 532)

NM_(—)015282-1786,gcgatctcgaagtgatatt (SEQ ID NO: 533)

NM_(—)015282-635,cagtcccggttgaatgtaa (SEQ ID NO: 534)

(Target Gene of RNAi)

NM_(—)006287, Homo sapiens tissue factor pathway inhibitor(lipoprotein-associated coagulation inhibitor) (TFPI).

(Target Sequences)

NM_(—)006287-225,ctcgacagtgcgaagaatt (SEQ ID NO: 535)

NM_(—)006287-227,cgacagtgcgaagaattta (SEQ ID NO: 536)

NM_(—)006287-228,gacagtgcgaagaatttat (SEQ ID NO: 537)

NM_(—)006287-230,cagtgcgaagaatttatat (SEQ ID NO: 538)

NM_(—)006287-393,gaatatgtcgaggttatat (SEQ ID NO: 539)

(Target Gene of RNAi)

NM_(—)004073, Homo sapiens cytokine-inducible kinase (CNK).

(Target Sequences)

NM_(—)004073-1283,gttgactactccaataagt (SEQ ID NO: 540)

NM_(—)004073-138,gcgcctacgctgtcaaagt (SEQ ID NO: 541)

NM_(—)004073-239,cgccacatcgtgcgttttt (SEQ ID NO: 542)

NM_(—)004073-1281,gggttgactactccaataa (SEQ ID NO: 543)

(Target Sequences Effective for Mouse Homolog)

NM_(—)004073-192,gcgagaagatcctaaatga (SEQ ID NO: 544)

NM_(—)004073-183,cgcatcagcgcgagaagat (SEQ ID NO: 545)

NM_(—)004073-190,gcgcgagaagatcctaaat (SEQ ID NO: 546)

(Target Gene of RNAi)

NM_(—)003384, Homo sapiens vaccinia related kinase 1 (VRK1).

(Target Sequences)

NM_(—)003384-776,ccttgggaggataatttga (SEQ ID NO: 547)

NM_(—)003384-773,cttccttgggaggataatt (SEQ ID NO: 548)

NM_(—)003384-195,caccttgtgttgtaaaagt (SEQ ID NO: 549)

NM_(—)003384-777,cttgggaggataatttgaa (SEQ ID NO: 550)

(Target Sequences Effective for Mouse Homolog)

NM_(—)003384-372,gttacaggtttatgataat (SEQ ID NO: 551)

NM_(—)003384-463,gcagctaagcttaagaatt (SEQ ID NO: 552)

NM_(—)003384-977,ggactaaaagctataggaa (SEQ ID NO: 553)

(Target Gene of RNAi)

NM_(—)006296, Homo sapiens vaccinia related kinase 2 (VRK2).

(Target Sequences)

NM_(—)006296-366,gactaggaatagatttaca (SEQ ID NO: 554)

NM_(—)006296-165,caagacatgtagtaaaagt (SEQ ID NO: 555)

NM_(—)006296-874,ggtatgtgctcatagttta (SEQ ID NO: 556)

NM_(—)006296-541,ggtttatcttgcagattat (SEQ ID NO: 557)

NM_(—)006296-113,ggatttggattgatatatt (SEQ ID NO: 558)

(Target Sequences Effective for Mouse Homolog)

NM_(—)006296-560,ggactttcctacagatatt (SEQ ID NO: 559)

NM_(—)006296-626,cataatgggacaatagagt (SEQ ID NO: 560)

NM_(—)006296-568,ctacagatattgtcccaat (SEQ ID NO: 561)

(Target Gene of RNAi)

NM_(—)004672, Homo sapiens mitogen-activated protein kinase kinasekinase 6 (MAP3K6).

(Target Sequences)

NM_(—)004672-2221,ctttctcctccgaactttt (SEQ ID NO: 562)

NM_(—)004672-1489,gatgttggagtttgattat (SEQ ID NO: 563)

NM_(—)004672-814,caaagagctccggctaata (SEQ ID NO: 564)

NM_(—)004672-51,ccctgcgggaggatgtttt (SEQ ID NO: 565)

NM_(—)004672-503,gccgagcagcataatgtct (SEQ ID NO: 566)

(Target Sequences Effective for Mouse Homolog)

NM_(—)004672-442,ggactactcggccatcatt (SEQ ID NO: 567)

NM_(—)004672-277,ctatttccgggagaccatt (SEQ ID NO: 568)

NM_(—)004672-1929,ggctgctcaagatttctga (SEQ ID NO: 569)

(Target Gene of RNAi)

NM_(—)005923, Homo sapiens mitogen-activated protein kinase kinasekinase 5 (MAP3K5).

(Target Sequences)

NM_(—)005923-3294,gatccactgaccgaaaaat (SEQ ID NO: 570)

NM_(—)005923-838,caggaaagctcgtaattta (SEQ ID NO: 571)

NM_(—)005923-840,ggaaagctcgtaatttata (SEQ ID NO: 572)

NM_(—)005923-1525,gtacctcaagtctattgta (SEQ ID NO: 573)

NM_(—)005923-2517,ctggtaccctccagtatat (SEQ ID NO: 574)

(Target Gene of RNAi)

NM_(—)020998, Homo sapiens macrophage stimulating 1 (hepatocyte growthfactor-like) (MST1).

(Target Sequences)

NM_(—)020998-943,ccgatttacgccagaaaaa (SEQ ID NO: 575)

NM_(—)020998-944,cgatttacgccagaaaaat (SEQ ID NO: 576)

NM_(—)020998-945,gatttacgccagaaaaata (SEQ ID NO: 577)

NM_(—)020998-698,ggtctggacgacaactatt (SEQ ID NO: 578)

NM_(—)020998-1827,ccaaaggtacgggtaatga (SEQ ID NO: 579)

(Target Gene of RNAi)

NM_(—)003576, Homo sapiens serine/threonine kinase 24 (STE20 homolog,yeast) (STK24).

(Target Sequences)

NM_(—)003576-348,gctccgcactagatctatt (SEQ ID NO: 580)

NM_(—)003576-349,ctccgcactagatctatta (SEQ ID NO: 581)

NM_(—)003576-351,ccgcactagatctattaga (SEQ ID NO: 582)

NM_(—)003576-352,cgcactagatctattagaa (SEQ ID NO: 583)

NM_(—)003576-437,ctccattcggagaagaaaa (SEQ ID NO: 584)

(Target Sequence Effective for Mouse Homolog)

NM_(—)003576-148,gttcaaaggcattgacaat (SEQ ID NO: 585)

(Target Gene of RNAi)

NM_(—)016542, Homo sapiens Mst3 and SOK1-related kinase (MST4).

(Target Sequences)

NM_(—)016542-857,ctgatagatcgttttaaga (SEQ ID NO: 586)

NM_(—)016542-139,gcaagtcgttgctattaaa (SEQ ID NO: 587)

NM_(—)016542-1133,gaagaactcgagaaaagta (SEQ ID NO: 588)

NM_(—)016542-556,ggctcctgaagttattcaa (SEQ ID NO: 589)

(Target Sequences Effective for Mouse Homolog)

NM_(—)016542-613,gggaattactgctattgaa (SEQ ID NO: 590)

NM_(—)016542-669,caatgagagttctgtttct (SEQ ID NO: 591)

NM_(—)016542-1063,gataatcacacctgcattt (SEQ ID NO: 592)

(Target Gene of RNAi)

NM_(—)002576, Homo sapiens p21/Cdc42/Rac1-activated kinase 1 (STE20homolog, yeast) (PAK1).

(Target Sequences)

NM_(—)002576-38,gcccctccgatgagaaata (SEQ ID NO: 593)

NM_(—)002576-788,ggcgatcctaagaagaaat (SEQ ID NO: 594)

NM_(—)002576-3,caaataacggcctagacat (SEQ ID NO: 595)

NM_(—)002576-154,ccgattttaccgatccatt (SEQ ID NO: 596)

(Target Sequences Effective for Mouse Homolog)

NM_(—)002576-1020,gggttgttatggaatactt (SEQ ID NO: 597)

NM_(—)002576-1165,catcaagagtgacaatatt (SEQ ID NO: 598).

NM_(—)002576-1015,gctgtgggttgttatggaa (SEQ ID NO: 599)

(Target Gene of RNAi)

NM_(—)002577, Homo sapiens p21 (CDKNlA)-activated kinase 2 (PAK2).

(Target Sequences)

NM_(—)002577-721,cataggtgaccctaagaaa (SEQ ID NO: 600)

NM_(—)002577-908,cccaacatcgttaactttt (SEQ ID NO: 601)

NM_(—)002577-909,ccaacatcgttaacttttt (SEQ ID NO: 602)

NM_(—)002577-557,ccggatcatacgaaatcaa (SEQ ID NO: 603)

NM_(—)002577-558,cggatcatacgaaatcaat (SEQ ID NO: 604)

(Target Gene of RNAi)

NM_(—)002578, Homo sapiens p21 (CDKNlA)-activated kinase 3 (PAK3).

(Target Sequences)

NM_(—)002578-458,catccttcgagtacaaaaa (SEQ ID NO: 605)

NM_(—)002578-1467,ctgtattccgtgacttttt (SEQ ID NO: 606)

NM_(—)002578-1469,gtattccgtgactttttaa (SEQ ID NO: 607)

NM_(—)002578-706,cacagatcggcaaagaaaa (SEQ ID NO: 608)

NM_(—)002578-3,ctgacggtctggataatga (SEQ ID NO: 609)

(Target Sequences Effective for Mouse Homolog)

NM_(—)002578-1376,cccccttaccttaatgaaa (SEQ ID NO: 610)

NM_(—)002578-219,cagactttgagcatacgat (SEQ ID NO: 611)

NM_(—)002578-254,gcagtcaccggggaattca (SEQ ID NO: 612)

(Target Gene of RNAi)

NM_(—)005884, Homo sapiens p21(CDKN1A)-activated kinase 4 (PAK4).

(Target Sequences)

NM_(—)005884-1502,gggataatggtgattgaga (SEQ ID NO: 613)

NM_(—)005884-1503,ggataatggtgattgagat (SEQ ID NO: 614)

NM_(—)005884-883,gccacagcgagtatcccat (SEQ ID NO: 615)

NM_(—)005884-77,cagcacgagcagaagttca (SEQ ID NO: 616)

NM_(—)005884-1494,ggtcgctggggataatggt (SEQ ID NO: 617)

(Target Gene of RNAi)

NM_(—)002755, Homo sapiens mitogen-activated protein kinase kinase 1(MAP2K1).

(Target Sequences)

NM_(—)002755-280,ggccagaaagctaattcat (SEQ ID NO: 618)

NM_(—)002755-402,gcgatggcgagatcagtat (SEQ ID NO: 619)

NM_(—)002755-404,gatggcgagatcagtatct (SEQ ID NO: 620)

NM_(—)002755-682,ctacatgtcgccagaaaga (SEQ ID NO: 621)

NM_(—)002755-1128,ccaccatcggccttaacca (SEQ ID NO: 622)

(Target Sequences Effective for Mouse Homolog)

NM_(—)002755-912,gacctcccatggcaatttt (SEQ ID NO: 623)

NM_(—)002755-915,ctcccatggcaatttttga (SEQ ID NO: 624)

NM_(—)002755-911,cgacctcccatggcaattt (SEQ ID NO: 625)

(Target Gene of RNAi)

NM_(—)030662, Homo sapiens mitogen-activated protein kinase kinase 2(MAP2K2).

(Target Sequences)

NM_(—)030662-1136,gccggctggttgtgtaaaa (SEQ ID No: 626)

NM_(—)030662-184,caaggtcggcgaactcaaa (SEQ ID NO: 627)

NM_(—)030662-959,ctcctggactatattgtga (SEQ ID NO: 628)

NM_(—)030662-183,ccaaggtcggcgaactcaa (SEQ ID NO: 629)

NM_(—)030662-711,ggttgcagggcacacatta (SEQ ID NO: 630)

(Target Gene of RNAi)

NM_(—)002756, Homo sapiens mitogen-activated protein kinase kinase 3(MAP2K3).

(Target Sequences)

NM_(—)002756-257,cgcacggtcgactgtttct (SEQ ID NO: 631)

NM_(—)002756-258,gcacggtcgactgtttcta (SEQ ID NO: 632)

NM_(—)002756-289,ctacggggcactattcaga (SEQ ID NO: 633)

NM_(—)002756-285,ccttctacggggcactatt (SEQ ID NO: 634)

NM_(—)002756-44,gactcccggaccttcatca (SEQ ID NO: 635)

(Target Sequences Effective for Mouse Homolog)

NM_(—)002756-129,gagcctatggggtggtaga (SEQ ID NO: 636)

NM_(—)002756-41,ctggactcccggaccttca (SEQ ID NO: 637)

NM_(—)002756-89,gaggctgatgacttggtga (SEQ ID NO: 638)

(Target Gene of RNAi)

NM_(—)002758, Homo sapiens mitogen-activated protein kinase kinase 6(MAP2K6).

(Target Sequences)

NM_(—)002758-394,ggatacatcactagataaa (SEQ ID NO: 639)

NM_(—)002758-395,gatacatcactagataaat (SEQ ID NO: 640)

NM_(—)002758-755,cttcgatttccctatgatt (SEQ ID NO: 641)

NM_(—)002758-340,cttttatggcgcactgttt (SEQ ID NO: 642)

NM_(—)002758-399,catcactagataaattcta (SEQ ID NO: 643)

(Target Sequences Effective for Mouse Homolog)

NM_(—)002758-312,ggacggtggactgtccatt (SEQ ID NO: 644)

NM_(—)002758-418,caaacaagttattgataaa (SEQ ID NO: 645)

NM_(—)002758-415,ctacaaacaagttattgat (SEQ ID NO: 646)

(Target Gene of RNAi)

NM_(—)003010, Homo sapiens mitogen-activated protein kinase kinase 4(MAP2K4).

(Target Sequences)

NM_(—)003010-543,ctacctcgtttgataagtt (SEQ ID NO: 647)

NM_(—)003010-1130,gcatgctatgtttgtaaaa (SEQ ID NO: 648)

NM_(—)003010-1056,ccaaaaggccaaagtataa (SEQ ID NO: 649)

(Target Sequences Effective for Mouse Homolog)

NM_(—)003010-1129,cgcatgctatgtttgtaaa (SEQ ID NO: 650)

NM_(—)003010-1057,caaaaggccaaagtataaa (SEQ ID NO: 651)

NM_(—)003010-452,gtaatgcggagtagtgatt (SEQ ID NO: 652)

(Target Gene of RNAi)

NM_(—)016123, Homo sapiens interleukin-1 receptor-associated kinase 4(IRAK4).

(Target Sequences)

NM_(—)016123-1299,gccaatgtcggcatgaaaa (SEQ ID NO: 653)

NM_(—)016123-1073,gctttgcgtggagaaataa (SEQ ID NO: 654)

NM_(—)016123-38,ctcaatgttggactaatta (SEQ ID NO: 655)

NM_(—)016123-769,cctctgcttagtatatgtt (SEQ ID NO: 656)

NM_(—)016123-1180,gttattgctagatattaaa (SEQ ID NO: 657)

(Target Gene of RNAi)

NM_(—)002880, Homo sapiens v-raf-1 murine leukemia viral oncogenehomolog 1 (RAF1).

(Target Sequences)

NM_(—)002880-1703,gatcttagtaagctatata (SEQ ID NO: 658)

NM_(—)002880-232,gcatgactgccttatgaaa (SEQ ID NO: 659)

NM_(—)002880-1597,ctatggcatcgtattgtat (SEQ ID NO: 660)

NM_(—)002880-1706,cttagtaagctatataaga (SEQ ID NO: 661)

NM_(—)002880-568,cagacaactcttattgttt (SEQ ID NO: 662)

(Target Gene of RNAi)

NM_(—)000020, Homo sapiens activin A receptor type II-like 1 (ACVRL1).

(Target Sequences)

NM_(—)000020-1453,caagaagacactacaaaaa (SEQ ID NO: 663)

NM_(—)000020-722,gagactgagatctataaca (SEQ ID NO: 664)

NM_(—)000020-1456,gaagacactacaaaaaatt (SEQ ID NO: 665)

NM_(—)000020-728,gagatctataacacagtat (SEQ ID NO: 666)

NM_(—)000020-846,gctccctctacgactttct (SEQ ID NO: 667)

(Target Gene of RNAi)

NM_(—)001105, Homo sapiens activin A receptor, type I (ACVR1).

(Target Sequences)

NM_(—)001105-1456,cacagcactgcgtatcaaa (SEQ ID NO: 668)

NM_(—)001105-428,gttgctctccgaaaattta (SEQ ID NO: 669)

NM_(—)001105-431,gctctccgaaaatttaaaa (SEQ ID NO: 670)

NM_(—)001105-1460,gcactgcgtatcaaaaaga (SEQ ID NO: 671)

NM_(—)001105-1458,cagcactgcgtatcaaaaa (SEQ ID NO: 672)

(Target Sequences Effective for Mouse Homolog)

NM_(—)001105-1306,caatgacccaagttttgaa (SEQ ID NO: 673)

NM_(—)001105-1381,gttctcagacccgacatta (SEQ ID NO: 674)

NM_(—)001105-281,caaggggactggtgtaaca (SEQ ID NO: 675)

(Target Gene of RNAi)

NM_(—)004302, Homo sapiens activin A receptor, type IB (ACVR1B).

(Target Sequences)

NM_(—)004302-609,cccgaaccatcgttttaca (SEQ ID NO: 676)

NM_(—)004302-610,ccgaaccatcgttttacaa (SEQ ID NO: 677)

NM_(—)004302-897,caattgaggggatgattaa (SEQ ID NO: 678)

NM_(—)004302-857,cacgggtccctgtttgatt (SEQ ID NO: 679)

NM_(—)004302-859,cgggtccctgtttgattat (SEQ ID NO: 680)

(Target Sequences Effective for Mouse Homolog)

NM_(—)004302-1119,gggtggggaccaaacgata (SEQ ID NO: 681)

NM_(—)004302-1063,cctggctgtccgtcatgat (SEQ ID NO: 682)

NM_(—)004302-1121,gtggggaccaaacgataca (SEQ ID NO: 683)

(Target Gene of RNAi)

NM_(—)145259, Homo sapiens activin A receptor, type IC (ACVR1C).

(Target Sequences)

NM_(—)145259-1419,ctgctcttcgtattaagaa (SEQ ID NO: 684)

NM_(—)145259-956,gctcatcgagacataaaat (SEQ ID NO: 685)

NM_(—)145259-825,gctccttatatgactattt (SEQ ID NO: 686)

NM_(—)145259-959,catcgagacataaaatcaa (SEQ ID NO: 687)

NM_(—)145259-1237,gtaccaattgccttattat (SEQ ID NO: 688)

(Target Gene of RNAi)

NM_(—)004612, Homo sapiens transforming growth factor, beta receptor I(activin A receptor type II-like kinase, 53 kDa) (TGFBR1).

(Target Sequences)

NM_(—)004612-236,cgagataggccgtttgtat (SEQ ID NO: 689)

NM_(—)004612-1451,gcattgcggattaagaaaa (SEQ ID NO: 690)

NM_(—)004612-463,ccatcgagtgccaaatgaa (SEQ ID NO: 691)

NM_(—)004612-492,cattagatcgcccttttat (SEQ ID NO: 692)

NM_(—)004612-1449,cagcattgcggattaagaa (SEQ ID NO: 693)

(Target Sequences Effective for Mouse Homolog)

NM_(—)004612-829,gttggtgtcagattatcat (SEQ ID NO: 694)

NM_(—)004612-288,caacatattgctgcaatca (SEQ ID NO: 695)

NM_(—)004612-839,gattatcatgagcatggat (SEQ ID NO: 696)

(Target Gene of RNAi)

NM_(—)004836, Homo sapiens eukaryotic translation initiation factor2-alpha kinase 3 (EIF2AK3).

(Target Sequences)

NM_(—)004836-1594,catagcaacaacgtttatt (SEQ ID NO: 697)

NM_(—)004836-1419,catatgataatggttatta (SEQ ID NO: 698)

NM_(—)004836-1900,ggtaatgcgagaagttaaa (SEQ ID NO: 699)

NM_(—)004836-1248,ctaatgaaaacgcaattat (SEQ ID NO: 700)

(Target Sequences Effective for Mouse Homolog)

NM_(—)004836-784,ctttgaacttcggtatatt (SEQ ID NO: 701)

NM_(—)004836-782,cactttgaacttcggtata (SEQ ID NO: 702)

NM_(—)004836-983,gaatgggagtaccagtttt (SEQ ID NO: 703)

(Target Gene of RNAi)

NM_(—)001433, Homo sapiens ER to nucleus signalling 1 (ERN1).

(Target Sequences)

NM_(—)001433-2407,cattgcacgagaattgata (SEQ ID NO: 704)

NM_(—)001433-2277,caggctgcgtcttttacta (SEQ ID NO: 705)

NM_(—)001433-2530,cgtgagcgacagaatagaa (SEQ ID NO: 706)

NM_(—)001433-1149,ccaaacatcgggaaaatgt (SEQ ID NO: 707)

NM_(—)001433-364,ggacatctggtatgttatt (SEQ ID NO: 708)

(Target Sequences Effective for Mouse Homolog)

NM_(—)001433-319,cccatgccgaagttcagat (SEQ ID NO: 709)

NM_(—)001433-2254,ctacacggtggacatcttt (SEQ ID NO: 710)

NM_(—)001433-324,gccgaagttcagatggaat (SEQ ID NO: 711)

(Target Gene of RNAi)

NM_(—)001278, Homo sapiens conserved helix-loop-helix ubiquitous kinase(CHUK).

(Target Sequences)

NM_(—)001278-746,ggagaagttcggtttagta (SEQ ID NO: 712)

NM_(—)001278-1879,ggccctcagtaatatcaaa (SEQ ID NO: 713)

NM_(—)001278-864,gacctgttgaccttacttt (SEQ ID NO: 714)

NM_(—)001278-2150,ggccatttaagcactatta (SEQ ID NO: 715)

NM_(—)001278-2151,gccatttaagcactattat (SEQ ID NO: 716)

(Target Sequences Effective for Mouse Homolog)

NM_(—)001278-645,ctggatataggcctttttt (SEQ ID NO: 717)

NM_(—)001278-1354,gttaagtcttcttagatat (SEQ ID NO: 718)

NM_(—)001278-1203,gtttatctgattgtgtaaa (SEQ ID NO: 719)

(Target Gene of RNAi)

NM_(—)014002, Homo sapiens inhibitor of kappa light polypeptide geneenhancer in B-cells, kinase epsilon (IKBKE).

(Target Sequences)

NM_(—)014002-2107,catcgaacggctaaataga (SEQ ID NO: 720)

NM_(—)014002-1724,ctggataaggtgaatttca (SEQ ID NO: 721)

NM_(—)014002-535,cctgcatcccgacatgtat (SEQ ID NO: 722)

NM_(—)014002-1220,ctgcaggcggattacaaca (SEQ ID NO: 723)

NM_(—)014002-1726,ggataaggtgaatttcagt (SEQ ID NO: 724)

(Target Sequence Effective for Mouse Homolog)

NM_(—)014002-54,ccactgccagtgtgtacaa (SEQ ID NO: 725)

(Target Gene of RNAi)

NM_(—)003177, Homo sapiens spleen tyrosine kinase (SYK).

(Target Sequences)

NM_(—)003177-1222,caatgaccccgctcttaaa (SEQ ID NO: 726)

NM_(—)003177-713,cagctagtcgagcattatt (SEQ ID NO: 727)

NM_(—)003177-849,ggtcagcgggtggaataat (SEQ ID NO: 728)

NM_(—)003177-715,gctagtcgagcattattct (SEQ ID NO: 729)

NM_(—)003177-1389,gacatgtcaaggataagaa (SEQ ID NO: 730)

(Target Sequences Effective for Mouse Homolog)

NM_(—)003177-1559,gctgatgaaaactactaca (SEQ ID NO: 731)

NM_(—)003177-1028,gacacagaggtgtacgaga (SEQ ID NO: 732)

NM_(—)003177-1560,ctgatgaaaactactacaa (SEQ ID NO: 733)

(Target Gene of RNAi)

NM_(—)153831, Homo sapiens PTK2 protein tyrosine kinase 2 (PTK2).

(Target Sequences)

NM_(—)153831-451,gaagagcgattatatgtta (SEQ ID NO: 734)

NM_(—)153831-1889,gtaatcggtcgaattgaaa (SEQ ID NO: 735)

NM_(—)153831-93,caatggagcgagtattaaa (SEQ ID NO: 736)

NM_(—)153831-2747,ctggaccggtcgaatgata (SEQ ID NO: 737)

NM_(—)153831-92,gcaatggagcgagtattaa (SEQ ID NO: 738)

(Target Sequences Effective for Mouse Homolog)

NM_(—)153831-1767,ctccagagtcaatcaattt (SEQ ID NO: 739)

NM_(—)153831-1766,gctccagagtcaatcaatt (SEQ ID NO: 740)

NM_(—)153831-599,gttggtttaaagcgatttt (SEQ ID NO: 741)

(Target Gene of RNAi)

NM_(—)173174, Homo sapiens PTK2B protein tyrosine kinase 2 beta (PTK2B).

(Target Sequences)

NM_(—)173174-1273,ggtcctgaatcgtattctt (SEQ ID NO: 742)

NM_(—)173174-1776,ccccagagtccattaactt (SEQ ID NO: 743)

NM_(—)173174-1723,ggacgaggactattacaaa (SEQ ID NO: 744)

NM_(—)173174-2486,gaccccatggtttatatga (SEQ ID NO: 745)

(Target Sequences Effective for Mouse Homolog)

NM_(—)173174-378,ggaggtatgaccttcaaat (SEQ ID NO: 746)

NM_(—)173174-1182,gcagcatagagtcagacat (SEQ ID NO: 747)

NM_(—)173174-376,gtggaggtatgaccttcaa (SEQ ID NO: 748)

(Target Gene of RNAi)

NM_(—)002944, Homo sapiens v-ros UR2 sarcoma virus oncogene homolog 1(avian) (ROS1).

(Target Sequences)

NM_(—)002944-417,gaagctggacttatactaa (SEQ ID NO: 749)

NM_(—)002944-2123,gacatggattggtataaca (SEQ ID NO: 750)

NM_(—)002944-2163,cgaaaggcgacgtttttgt (SEQ ID NO: 751)

NM_(—)002944-1385,caagccaagcgaatcattt (SEQ ID NO: 752)

NM_(—)002944-416,ggaagctggacttatacta (SEQ ID NO: 753)

(Target Sequences Effective for Mouse Homolog)

NM_(—)002944-3048,ctgtcactccttataccta (SEQ ID NO: 754)

NM_(—)002944-3044,ctttctgtcactccttata (SEQ ID NO: 755)

NM_(—)002944-1051,caacatgtctgatgtatct (SEQ ID NO: 756)

(Target Gene of RNAi)

NM_(—)004304, Homo sapiens anaplastic lymphoma kinase (Ki-1) (ALK).

(Target Sequences)

NM_(—)004304-2469,ccacctacgtatttaagat (SEQ ID NO: 757)

NM_(—)004304-4067,cctgtataccggataatga (SEQ ID NO: 758).

NM_(—)004304-2468,gccacctacgtatttaaga (SEQ ID NO: 759)

NM_(—)004304-4183,cgctttgccgatagaatat (SEQ ID NO: 760)

NM_(—)004304-2922,gccacggggaagtgaatat (SEQ ID NO: 761)

(Target Sequences Effective for Mouse Homolog)

NM_(—)004304-3258,ccatcatgaccgactacaa (SEQ ID NO: 762)

NM_(—)004304-2833,caatgaccccgaaatggat (SEQ ID NO: 763)

NM_(—)004304-3156,ccggcatcatgattgtgta (SEQ ID NO: 764)

(Target Gene of RNAi)

NM_(—)000245, Homo sapiens met proto-oncogene (hepatocyte growth factorreceptor) (MET).

(Target Sequences)

NM_(—)000245-2761,gaacagcgagctaaatata (SEQ ID NO: 765)

NM_(—)000245-1271,cagcgcgttgacttattca (SEQ ID NO: 766)

NM_(—)000245-1086,gtgcattccctatcaaata (SEQ ID NO: 767)

NM_(—)000245-725,gattcttaccccattaagt (SEQ ID NO: 768)

NM_(—)000245-3619,caaagcgatgaaatatctt (SEQ ID NO: 769)

(Target Sequences Effective for Mouse Homolog)

NM_(—)000245-2987,catttggataggcttgtaa (SEQ ID NO: 770)

NM_(—)000245-801,ctctagatgctcagacttt (SEQ ID NO: 771)

NM_(—)000245-2660,gttaaaggtgaagtgttaa (SEQ ID NO: 772)

(Target Gene of RNAi)

NM_(—)002529, Homo sapiens neurotrophic tyrosine kinase, receptor, type1 (NTRK1).

(Target Sequences)

NM_(—)002529-2091,gcatcctgtaccgtaagtt (SEQ ID NO: 773)

NM_(—)002529-345,ggctcagtcgcctgaatct (SEQ ID NO: 774)

NM_(—)002529-347,ctcagtcgcctgaatctct (SEQ ID NO: 775)

NM_(—)002529-953,ggctccgtgctcaatgaga (SEQ ID NO: 776)

NM_(—)002529-1987,ggtcaagattggtgatttt (SEQ ID NO: 777)

(Target Gene of RNAi)

NM_(—)006180, Homo sapiens neurotrophic tyrosine kinase, receptor, type2 (NTRK2).

(Target Sequences)

NM_(—)006180-358,caattttacccgaaacaaa (SEQ ID NO: 778)

NM_(—)006180-1642,catcaagcgacataacatt (SEQ ID NO: 779)

NM_(—)006180-663,gtgatccggttcctaatat (SEQ ID NO: 780)

NM_(—)006180-665,gatccggttcctaatatgt (SEQ ID NO: 781)

NM_(—)006180-792,cttgtgtggcggaaaatct (SEQ ID NO: 782)

(Target Sequences Effective for Mouse Homolog)

NM_(—)006180-562,cctgcagatacccaattgt (SEQ ID NO: 783)

NM_(—)006180-898,ctggtgcattccattcact (SEQ ID NO: 784)

NM_(—)006180-735,cacagggctccttaaggat (SEQ ID NO: 785)

(Target Gene of RNAi)

NM_(—)000208, Homo sapiens insulin receptor (INSR).

(Target Sequences)

NM_(—)000208-2562,gccctgtgacgcatgaaat (SEQ ID NO: 786)

NM_(—)000208-2565,ctgtgacgcatgaaatctt (SEQ ID NO: 787)

NM_(—)000208-3492,gcatggtcgcccatgattt (SEQ ID NO: 788)

NM_(—)000208-3493,catggtcgcccatgatttt (SEQ ID NO: 789)

NM_(—)000208-329,ggatcacgactgttcttta (SEQ ID NO: 790)

(Target Sequences Effective for Mouse Homolog)

NM_(—)000208-2911,gattggaagtatttatcta (SEQ ID NO: 791)

NM_(—)000208-902,caccaatacgtcattcaca (SEQ ID NO: 792)

NM_(—)000208-1514,cggacatcttttgacaaga (SEQ ID NO: 793)

(Target Gene of RNAi)

NM_(—)000323, Homo sapiens ret proto-oncogene (multiple endocrineneoplasia and medullary thyroid carcinoma 1, Hirschsprung disease)(RET).

(Target Sequences)

NM_(—)000323-2679,gcttgtcccgagatgttta (SEQ ID NO: 794)

NM_(—)000323-3066,catctgactccctgattta (SEQ ID NO: 795)

NM_(—)000323-3069,ctgactccctgatttatga (SEQ ID NO: 796)

NM_(—)000323-2680,cttgtcccgagatgtttat (SEQ ID NO: 797)

NM_(—)000323-2728,gggtcggattccagttaaa (SEQ ID NO: 798)

(Target Sequences Effective for Mouse Homolog)

NM_(—)000323-3159,ccacatggattgaaaacaa (SEQ ID NO: 799)

NM_(—)000323-3156,cttccacatggattgaaaa (SEQ ID NO: 800)

NM_(—)000323-3155,ccttccacatggattgaaa (SEQ ID NO: 801)

(Target Gene of RNAi)

NM_(—)006293, Homo sapiens TYRO3 protein tyrosine kinase (TYRO3).

(Target Sequences)

NM_(—)006293-1494,gcatcagcgatgaactaaa (SEQ ID NO: 802)

NM_(—)006293-2207,gaaaacgctgagatttaca (SEQ ID NO: 803)

NM_(—)006293-2394,gccaggaccccttatacat (SEQ ID NO: 804)

NM_(—)006293-2399,gaccccttatacatcaaca (SEQ ID NO: 805)

NM_(—)006293-1493,ggcatcagcgatgaactaa (SEQ ID NO: 806)

(Target Gene of RNAi)

NM_(—)182925, Homo sapiens fins-related tyrosine kinase 4 (FLT4).

(Target Sequences)

NM_(—)182925-758,gtgtgggctgagtttaact (SEQ ID NO: 807)

NM_(—)182925-756,ccgtgtgggctgagtttaa (SEQ ID NO: 808)

NM_(—)182925-1217,ggcctgaggcgcaacatca (SEQ ID NO: 809)

NM_(—)182925-1827,gcaagaacgtgcatctgtt (SEQ ID NO: 810)

NM_(—)182925-908,gacctgggctcgtatgtgt (SEQ ID NO: 811)

(Target Sequences Effective for Mouse Homolog)

NM_(—)182925-2033,cggctcacgcagaacttga (SEQ ID NO: 812)

NM_(—)182925-330,gctactacaagtacatcaa (SEQ ID NO: 813)

(Target Gene of RNAi)

NM_(—)004119, Homo sapiens fins-related tyrosine kinase 3 (FLT3).

(Target Sequences)

NM_(—)004119-1569,gtgagacgatccttttaaa (SEQ ID NO: 814)

NM_(—)004119-2490,gattggctcgagatatcat (SEQ ID NO: 815)

NM_(—)004119-1571,gagacgatccttttaaact (SEQ ID NO: 816)

NM_(—)004119-32,ccgctgctcgttgtttttt (SEQ ID NO: 817)

NM_(—)004119-730,gttcacaatagatctaaat (SEQ ID NO: 818)

(Target Sequences Effective for Mouse Homolog)

NM_(—)004119-92,gtgatcaagtgtgttttaa (SEQ ID NO: 819)

NM_(—)004119-1483,ggtgtcgagcagtactcta (SEQ ID NO: 820)

NM_(—)004119-1456,ggctaacagaaaagtgttt (SEQ ID NO: 821)

(Target Gene of RNAi)

NM_(—)002253, Homo sapiens kinase insert domain receptor (a type IIIreceptor tyrosine kinase) (KDR).

(Target Sequences)

NM_(—)002253-617,gaaagttaccagtctatta (SEQ ID NO: 822)

NM_(—)002253-865,gagcaccttaactatagat (SEQ ID NO: 823)

NM_(—)002253-2020,gaatcagacgacaagtatt (SEQ ID NO: 824)

NM_(—)002253-815,gtaaaccgagacctaaaaa (SEQ ID NO: 825)

NM_(—)002253-2586,ggacagtagcagtcaaaat (SEQ ID NO: 826)

(Target Sequences Effective for Mouse Homolog)

NM_(—)002253-3032,gtggctaagggcatggagt (SEQ ID NO: 827)

NM_(—)002253-3627,ccaaattccattatgacaa (SEQ ID NO: 828)

NM_(—)002253-3626,cccaaattccattatgaca (SEQ ID NO: 829)

(Target Gene of RNAi)

NM_(—)002609, Homo sapiens platelet-derived growth factor receptor, betapolypeptide (PDGFRB).

(Target Sequences)

NM_(—)002609-961,ggtgggcacactacaattt (SEQ ID NO: 830)

NM_(—)002609-2881,gttgggcgaaggttacaaa (SEQ ID NO: 831)

NM_(—)002609-409,ctttctcacggaaataact (SEQ ID NO: 832)

NM_(—)002609-278,gacacgggagaatactttt (SEQ ID NO: 833)

NM_(—)002609-3048,gtgacaacgactatatcat (SEQ ID NO: 834)

(Target Sequences Effective for Mouse Homolog)

NM_(—)002609-633,catccatcaacgtctctgt (SEQ ID NO: 835)

NM_(—)002609-2784,cctccgacgagatctatga (SEQ ID NO: 836)

(Target Gene of RNAi)

NM_(—)005433, Homo sapiens v-yes-1 Yamaguchi sarcoma viral oncogenehomolog 1 (YESI).

(Target Sequences)

NM_(—)005433-525,gaaatcaacgaggtatttt (SEQ ID NO: 837)

NM_(—)005433-670,cacaaccagagcacaattt (SEQ ID NO: 838)

NM_(—)005433-1333,gtatggtcggtttacaata (SEQ ID NO: 839)

NM_(—)005433-1331,ctgtatggtcggtttacaa (SEQ ID NO: 840)

NM_(—)005433-416,ggttatatcccgagcaatt (SEQ ID NO: 841)

(Target Sequences Effective for Mouse Homolog)

NM_(—)005433-953,caagaagctcagataatga (SEQ ID NO: 842)

NM_(—)005433-1,gggctgcattaaaagtaaa (SEQ ID NO: 843)

NM_(—)005433-4,ctgcattaaaagtaaagaa (SEQ ID NO: 844)

(Target Gene of RNAi)

NM_(—)002005, Homo sapiens feline sarcoma oncogene (FES).

(Target Sequences)

NM_(—)002005-1696,gattggacgggggaacttt (SEQ ID NO: 845)

NM_(—)002005-2181,cacctgaggcccttaacta (SEQ ID NO: 846)

NM_(—)002005-1553,ggctttcctagcattcctt (SEQ ID NO: 847)

NM_(—)002005-683,gaatacctggagattagca (SEQ ID NO: 848)

NM_(—)002005-74,ctactggagggcatgagaa (SEQ ID NO: 849)

(Target Gene of RNAi)

NM_(—)000633, Homo sapiens B-cell CLL/lymphoma 2 (BCL2).

(Target Sequences)

NM_(—)000633-43,gatgaagtacatccattat (SEQ ID NO: 850)

NM_(—)000633-41,gtgatgaagtacatccatt (SEQ ID NO: 851)

(Target Sequences Effective for Mouse Homolog)

NM_(—)000633-452,gagttcggtggggtcatgt (SEQ ID NO: 852)

NM_(—)000633-454,gttcggtggggtcatgtgt (SEQ ID NO: 853)

NM_(—)000633-525,ggatgactgagtacctgaa (SEQ ID NO: 854)

(Target Gene of RNAi)

NM_(—)001167, Homo sapiens baculoviral IAP repeat-containing 4 (BIRC4).

(Target Sequences)

NM_(—)001167-302,gccacgcagtctacaaatt (SEQ ID NO: 855)

NM_(—)001167-794,gaagcacggatctttactt (SEQ ID NO: 856)

NM_(—)001167-485,gaagaagctagattaaagt (SEQ ID NO: 857)

NM_(—)001167-402,cacatgcagactatctttt (SEQ ID NO: 858)

(Target Sequences Effective for Mouse Homolog)

NM_(—)001167-71,gaagagtttaatagattaa (SEQ ID NO: 859)

NM_(—)001167-68,gtagaagagtttaatagat (SEQ ID NO: 860)

NM_(—)001167-1354,ctgtatggatagaaatatt (SEQ ID NO: 861)

(Target Gene of RNAi)

NM_(—)139317, Homo sapiens baculoviral IAP repeat-containing 7 (livin)(BIRC7).

(Target Sequences)

NM_(—)139317-458,ctgctccggtcaaaaggaa (SEQ ID NO: 862)

NM_(—)139317-457,cctgctccggtcaaaagga (SEQ ID NO: 863)

NM_(—)139317-743,gagaggacgtgcaaggtgt (SEQ ID NO: 864)

NM_(—)139317-774,ccgtgtccatcgtctttgt (SEQ ID NO: 865)

NM_(—)139317-417,cctggacggagcatgccaa (SEQ ID NO: 866)

(Target Gene of RNAi)

NM_(—)005036, Homo sapiens peroxisome proliferative activated receptor,alpha (PPARA).

(Target Sequences)

NM_(—)005036-922,gctaaaatacggagtttat (SEQ ID NO: 867)

NM_(—)005036-1243,ccacccggacgatatcttt (SEQ ID NO: 868)

NM_(—)005036-711,cttttgtcatacatgatat (SEQ ID NO: 869)

NM_(—)005036-498,cacacaacgcgattcgttt (SEQ ID NO: 870)

NM_(—)005036-988,gctggtagcgtatggaaat (SEQ ID NO: 871)

(Target Gene of RNAi)

NM_(—)138712, Homo sapiens peroxisome proliferative activated receptor,gamma (PPARG).

(Target Sequences)

NM_(—)138712-953,ggagtccacgagatcattt (SEQ ID NO: 872)

NM_(—)138712-304,ctccctcatggcaattgaa (SEQ ID NO: 873)

NM_(—)138712-954,gagtccacgagatcattta (SEQ ID NO: 874)

NM_(—)138712-445,ctgtcggatccacaaaaaa (SEQ ID NO: 875)

NM_(—)138712-409,cagattgaagcttatctat (SEQ ID NO: 876)

(Target Sequences Effective for Mouse Homolog)

NM_(—)138712-239,gcatctccaccttattatt (SEQ ID NO: 877)

NM_(—)138712-688,ggcgagggcgatcttgaca (SEQ ID NO: 878)

NM_(—)138712-664,gtccttcccgctgaccaaa (SEQ ID NO: 879)

(Target Gene of RNAi)

NM_(—)004421, Homo sapiens dishevelled, dsh homolog 1 (Drosophila)(DVL1).

(Target Sequences)

NM_(—)004421-1173,ccgtcgtccgggtcatgca (SEQ ID NO: 880)

(Target Gene of RNAi)

NM_(—)004422, Homo sapiens dishevelled, dsh homolog 2 (Drosophila)(DVL2).

(Target Sequences)

NM_(—)004422-1253,gtccatacggacatggcat (SEQ ID NO: 881)

(Target Gene of RNAi)

NM_(—)004423, Homo sapiens dishevelled, dsh homolog 3 (Drosophila)(DVL3).

(Target Sequences)

NM_(—)004423-1197,gcctagacgacttccactt (SEQ ID NO: 882)

(Target Gene of RNAi)

NC_(—)001802, Human immunodeficiency virus 1, complete genome.

(Target Sequences)

NC_(—)001802-8242,ggacagatagggttataga (SEQ ID NO: 883)

NC_(—)001802-340,gcgagagcgtcagtattaa (SEQ ID NO: 884)

NC_(—)001802-1222,gtagaccggttctataaaa (SEQ ID NO: 885)

NC_(—)001802-1818,cgacccctcgtcacaataa (SEQ ID NO: 886)

NC_(—)001802-4973,gccctaggtgtgaatatca (SEQ ID NO: 887)

NC_(—)001802-5224,gcttagggcaacatatcta (SEQ ID NO: 888)

NC_(—)001802-550,gaagaacttagatcattat (SEQ ID NO: 889)

NC_(—)001802-1777,gaactgtatcctttaactt (SEQ ID NO: 890)

NC_(—)001802-3244,gaaagactcctaaatttaa (SEQ ID NO: 891)

NC_(—)001802-5225,cttagggcaacatatctat (SEQ ID NO: 892)

ADVANTAGES OF THE INVENTION

According to the present invention, siRNA actually having an RNAi Effectcan be obtained with high probability. Thus, when preparing novel siRNA,it is possible to greatly reduce the effort required to carry outrepeated tests of trial and error, based on the experiences of theresearcher, in actually synthesizing siRNA and in confirming whether thesynthesized product has an RNAi Effect. Namely, the present invention isextremely preferred for carrying out a search or for creation of siRNAhaving a novel sequence. Furthermore, by using the present invention, awide variety of desired siRNA can be obtained in a short time. Sincenecessity for actual preparation of siRNA in a trial-and-error mannerhas been reduced, it becomes possible to greatly reduce the costrequired for testing and manufacturing techniques, in which RNAinterference is used. Additionally, the present invention not onlygreatly simplifies all testing and manufacturing techniques, in whichthe RNAi Effect is used, but also significantly improves theirreliability as techniques. The present invention is particularlyEffective in performing RNA interference in higher animals such asmammals.

INDUSTRIAL APPLICABILITY

As described above, the present invention relates to RNA interferenceand more particularly, for example, to a method for designing sequencesof polynucleotides for causing RNA interference, the method improvingefficiency in testing, manufacturing, etc., in which RNA interference isused.

1. A method for searching a target base sequence of RNA interferencecomprising: searching a sequence segment, conforming to the followingrules (a) to (d), from the base sequences of a target gene of RNAinterference: (a) The 3′ end base is adenine, thymine, or uracil, (b)The 5′ end base is guanine or cytosine, (c) A 7-base sequence from the3′ end is rich in one or more types of bases selected from the groupconsisting of adenine, thymine, and uracil, and (d) The number of basesis within a range that allows RNA interference to occur without causingcytotoxicity.
 2. The method for searching the target base sequenceaccording to claim 1, wherein, in the rule (c), at least three basesamong the seven bases are one or more types of bases selected from thegroup consisting of adenine, thymine, and uracil.
 3. The method forsearching the target base sequence according to claim 1 or 2, wherein,in the rule (d), the number of bases is 13 to
 28. 4. A method fordesigning a base sequence of a polynucleotide for causing RNAinterference comprising: searching a base sequence, conforming to therules (a) to (d) below, from the base sequences of a target gene anddesigning a base sequence homologous to the searched base sequence: (a)The 3′ end base is adenine, thymine, or uracil, (b) The 5′ end base isguanine or cytosine, (c) A 7-base sequence from the 3′ end is rich inone or more types of bases selected from the group consisting ofadenine, thymine, and uracil, and (d) The number of bases is within arange that allows RNA interference to occur without causingcytotoxicity.
 5. The method for designing the base sequence according toclaim 4, wherein, in the rule (c), at least three bases among the sevenbases are one or more types of bases selected from the group consistingof adenine, thymine, and uracil.
 6. The method for designing the basesequence according to claim 4 or 5, wherein the number of bases in thehomologous base sequence designed is 13 to
 28. 7. The method fordesigning the base sequence according to any one of claims 4 to 6,wherein designing is performed so that at least 80% of bases in thehomologous base sequence designed corresponds to the base sequencesearched.
 8. The method for designing the base sequence according to anyone of claims 4 to 7, wherein the 3′ end base of the base sequencesearched is the same as the 3′ end base of the base sequence designed,and the 5′ end base of the base sequence searched is the same as the 5′end base of the base sequence designed.
 9. The method for designing thebase sequence according to any one of claims 4 to 8, wherein anoverhanging portion is added to the 3′ end of the polynucleotide.
 10. Amethod for producing a double-stranded polynucleotide comprising:forming one strand by providing an overhanging portion to the 3′ end ofa base sequence homologous to a prescribed sequence which is containedin the base sequences of a target gene and which conforms to thefollowing rules (a) to (d); and forming the other strand by providing anoverhanging portion to the 3′ end of a base sequence complementary tothe base sequence homologous to the prescribed sequence, wherein thenumber of bases in each strand is 15 to 30: (a) The 3′ end base isadenine, thymine, or uracil, (b) The 5′ end base is guanine or cytosine,(c) A 7-base sequence from the 3′ end is rich in one or more types ofbases selected from the group consisting of adenine, thymine, anduracil, and (d) The number of bases is within a range that allows RNAinterference to occur without causing cytotoxicity.
 11. Adouble-stranded polynucleotide synthesized by searching a sequencesegment having 13 to 28 bases, conforming to the following rules (a) to(d), from the base sequences of a target gene for RNA interference,forming one strand by providing an overhanging portion to the 3′-end ofa base sequence homologous to a prescribed sequence which is containedin the base sequences of the target gene and which conforms to thefollowing rules (a) to (d), and forming the other strand by providing anoverhanging portion to the 3′ end of a base sequence complementary tothe base sequence homologous to the prescribed sequence, wherein thenumber of bases in each strand is 15 to 30: (a) The 3′ end base isadenine, thymine, or uracil, (b) The 5′ end base is guanine or cytosine,(c) A 7-base sequence from the 3′ end is rich in one or more types ofbases selected from the group consisting of adenine, thymine, anduracil, and (d) The number of bases is within a range that allows RNAinterference to occur without causing cytotoxicity.
 12. A method forinhibiting gene expression comprising the steps of: searching a sequencesegment having 13 to 28 bases, conforming to the following rules (a) to(d), from the base sequences of a target gene for RNA interference;synthesizing a double-stranded polynucleotide such that one strand isformed by providing an overhanging portion to the 3′ end of a basesequence homologous to a prescribed sequence which is contained in thebase sequences of the target gene and which conforms to the followingrules (a) to (d), the other strand is formed by providing an overhangingportion to the 3′ end of a base sequence complementary to the basesequence homologous to the prescribed sequence, and the number of basesin each strand is 15 to 30; and introducing the synthesizeddouble-stranded polynucleotide into an expression system of the targetgene of which expression is to be inhibited to inhibit the expression ofthe target gene: (a) The 3′ end base is adenine, thymine, or uracil, (b)The 5′ end base is guanine or cytosine, (c) A 7-base sequence from the3′ end is rich in one or more types of bases selected from the groupconsisting of adenine, thymine, and uracil, and (d) The number of basesis within a range that allows RNA interference to occur without causingcytotoxicity.
 13. A base sequence processing apparatus characterized inthat it comprises: partial base sequence creation means for acquiringbase sequence information of a target gene for RNA interference andcreating partial base sequence information corresponding to a sequencesegment having a predetermined number of bases in the base sequenceinformation; 3′ end base determination means for determining whether the3′ end base in the partial base sequence information created by thepartial base sequence creation means is adenine, thymine, or uracil; 5′end base determination means for determining whether the 5′ end base inthe partial base sequence information created by the partial basesequence creation means is guanine or cytosine; predetermined baseinclusion determination means for determining whether base sequenceinformation comprising 7 bases at the 3′ end in the partial basesequence information created by the partial base sequence creation meansis rich in one or more types of bases selected from the group consistingof adenine, thymine, and uracil; and prescribed sequence selection meansfor selecting prescribed sequence information which specifically causesRNA interference in the target gene from the partial base sequenceinformation created by the partial base sequence creation means, basedon the results determined by the 3′-base determination means, the 5′ endbase determination means, and the predetermined base inclusiondetermination means.
 14. The base sequence processing apparatusaccording to claim 13, characterized in that the partial base sequencecreation means further comprises region-specific base sequence creationmeans for creating the partial base sequence information having thepredetermined number of bases from a segment corresponding to a codingregion or transcription region of the target gene in the base sequenceinformation.
 15. The base sequence processing apparatus according toclaim 13 or 14, characterized in that the partial base sequence creationmeans further comprises common base sequence creation means for creatingthe partial base sequence information having the predetermined number ofbases which is common in a plurality of base sequence informationderived from different organisms.
 16. The base sequence processingapparatus according to any one of claims 13 to 15, characterized in thatthe base sequence information that is rich corresponds to base sequenceinformation comprising the 7 bases containing at least 3 bases which areone or more types of bases selected from the group consisting ofadenine, thymine, and uracil.
 17. The base sequence processing apparatusaccording to any one of claims 13 to 16, wherein the predeterminednumber of bases is 13 to
 28. 18. The base sequence processing apparatusaccording to any one of claims 13 to 17, characterized in that thepartial base sequence creation means further comprises overhangingportion-containing base sequence creation means for creating the partialbase sequence information containing an overhanging portion.
 19. Thebase sequence processing apparatus according to any one of claims 13 to17, characterized in that it comprises: overhanging-portion additionmeans for adding an overhanging portion to at least one end of theprescribed sequence information.
 20. The base sequence processingapparatus according to claim 18 or 19, wherein the number of bases inthe overhanging portion is
 2. 21. The base sequence processing apparatusaccording to any one of claims 13 to 20, characterized in that itcomprises: identical/similar base sequence search means forsearching-base sequence information, identical or similar to theprescribed sequence information, from other base sequence information;and unrelated gene target evaluation means for evaluating whether theprescribed sequence information targets genes unrelated to the targetgene based on the identical or similar base sequence informationsearched by the identical/similar base sequence search means.
 22. Thebase sequence processing apparatus according to claim 21, characterizedin that the unrelated gene target evaluation means further comprises:total sum calculation means for calculating the total sum of reciprocalsof the values showing the degree of identity or similarity based on thetotal amount of base sequence information on the genes unrelated to thetarget gene in the identical or similar base sequence informationsearched by the identical/similar base sequence search means and thevalues showing the degree of identity or similarity attached to the basesequence information on the genes unrelated to the target gene; andtotal sum-based target evaluation means for evaluating whether theprescribed sequence information targets the genes unrelated to thetarget gene based on the total sum calculated by the total sumcalculation means.
 23. A program for running base sequence processingmethod on computer, characterized in that it comprises: a partial basesequence creation step of acquiring base sequence information of atarget gene for RNA interference and creating partial base sequenceinformation corresponding to a sequence segment having a predeterminednumber of bases in the base sequence information; a 3′ end basedetermination step of determining whether the 3′ end base in the partialbase sequence information created in the partial base sequence creationstep is adenine, thymine, or uracil; a 5′ end base determination step ofdetermining whether the 5′ end base in the partial base sequenceinformation created in the partial base sequence creation step isguanine or cytosine; a predetermined base inclusion determination stepof determining whether base sequence information comprising 7 bases atthe 3′ end in the partial base sequence information created in thepartial base sequence creation step is rich in one or more types ofbases selected from the group consisting of adenine, thymine, anduracil; and a prescribed sequence selection step of selecting, based onthe results determined in the 3′ base determination step, the 5′ endbase determination step, and the predetermined base inclusiondetermination step, prescribed sequence information which specificallycauses RNA interference in the target gene from the partial basesequence information created in the partial base sequence creation step.24. A computer-readable recording medium characterized in that theprogram according to claim 23 is recorded in the medium.
 25. A basesequence processing system which comprises a base sequence processingapparatus processing base sequence information of a target gene for RNAinterference and a client apparatus, the base sequence processingapparatus and the client apparatus being connected to each other via anetwork in a communicable manner, characterized in that the clientapparatus comprises: base sequence transmission means for transmitting aname of the target gene or the base sequence information to the basesequence processing apparatus; and prescribed sequence acquisition meansfor acquiring prescribed sequence information which is transmitted fromthe base sequence processing apparatus and which specifically causes RNAinterference in the target gene, and the base sequence processingapparatus comprises: partial base sequence creation means for acquiringbase sequence information corresponding to the name of the target geneor the base sequence information transmitted from the client apparatusand creating partial base sequence information corresponding to asequence segment having a predetermined number of bases in the basesequence information; 3′ end base determination means for determiningwhether the 3′ end base in the partial base sequence information createdby the partial base sequence creation means is adenine, thymine, oruracil; 5′ end base determination means for determining whether the 5′end base in the partial base sequence information created by the partialbase sequence creation means is guanine or cytosine; predetermined baseinclusion determination means for determining whether base sequenceinformation comprising 7 bases at the 3′ end in the partial basesequence information created by the partial base sequence creation meansis rich in one or more types of bases selected from the group consistingof adenine, thymine, and uracil; prescribed sequence selection means forselecting the prescribed sequence information from the partial basesequence information created by the partial base sequence creationmeans, based on the results determined by the 3′ base determinationmeans, the 5′ end base determination means, and the predetermined baseinclusion determination means; and prescribed sequence transmissionmeans for transmitting the prescribed sequence information selected bythe prescribed sequence selection means to the client apparatus.